Stromal vascular fraction: Mechanisms and application in reproductive disorders

Abstract

Stromal vascular fraction (SVF) is a complex mixture derived from adipose tissue, consisting of a variety of cells. Due to its potential for tissue repair, immunomodulation, and support of angiogenesis, SVF represents a promising frontier in regenerative medicine and offers potential therapy for a range of disease conditions. In this article, we delve into the mechanisms through which SVF exerts its effects and explore its potential applications in treating both male and female reproductive disorders, including erectile dysfunction, testicular injury, stress urinary incontinence and intrauterine adhesion.

Key Words: Stromal vascular fraction; Angiogenesis; Inflammation; Regenerative medicine; Reproductive disorders

Core Tip: This article will analyze the minireview by Jeyaraman et al, with a focus on the future direction of stromal vascular fraction (SVF) for reproductive therapy. SVF received increasing attention for its multi-faceted function including immunomodulation, pro-angiogenesis and tissue repair. By reviewing the current research in the field of erectile dysfunction, testicular injury, intrauterine adhesion, and stress urinary incontinence, we highlight the opportunities of SVF in treating reproductive disorders and improving infertility. With advancements in standardizing protocol of SVF isolation and more clinical trials exploring its efficacy and safety, SVF should be promoted for more applications in practice.

TO THE EDITOR

Stromal vascular fraction (SVF) is a complex mixture derived from adipose tissue, consisting of a variety of cell types including adipose-derived stem cells (ASC), endothelial progenitor cells, pericytes, fibroblasts, and immune cells[1]. It is advantaged by its rich sources, since adipose tissue can be accessed from many different sites such as the flank area and abdomen. Adipose tissue has around 40% higher yield of mesenchymal stem cells compared to umbilical cord blood[2]. The ASC can be isolated from SVF for application in regenerative medicine, after liposuction, lipid tissue processing, filtration, centrifugation, cell separation, cell culture and expansion, and characterization[3,4]. Compared to ASC, one significant strength of SVF is that it does not require complicated processing and culturing to be put into use, thus saving the time and cost associated with treatment preparation. Currently, SVF has a wide range of clinical applications, with osteoarthritis and other joint conditions being the most investigated. SVF has also been used for plastic surgery, and it is under clinical investigation for chronic wound healing and cardiovascular and respiratory diseases[4,5].

The minireview written by Jeyaraman et al[6] clearly presented the isolation stages and analyzed the detailed processes with their specific advantages and limitations. Selection between autologous and allogenic sources of SVF, different impacts of anesthesia choice, tissue extraction, aspiration, centrifugation, and delivery are included, providing researchers with a consolidated understanding of the technical aspects of ASC isolation. In summary, its efforts in analyzing the controlling variables can inspire critical thinking regarding the improvement of standardized protocols[6].

According to the statistics in the first decade of the 21^st century, the infertility rate ranges between 8% and 30% across different countries, with many regions of especially high infertility rates scattered in almost every continent. Meanwhile, there are emerging infertility issues that still do not have widely accepted treatment methods[7]. The extensive exploration of SVF application as regenerative medicine will be of great importance in finding the possibility of addressing infertility problems or using it as a supplementary treatment. In this article, we will focus on the mechanisms of SVF and its future directions in treating reproductive disorders.

MECHANISMS OF SVF

Immunomodulatory function

First, the SVF contains many cells that can modulate both innate and adaptive immune responses, such as T cells, B cells, T regulatory cells, macrophages, and natural killer cells, though these immune cells are at relatively low levels[8]. For example, the T regulatory cells can maintain immune homeostasis as they modulate the T cell activation and proliferation and then provide tolerance. Another example would be the M2 subtype of macrophage, as it has anti-inflammatory effects[9,10]. Second, SVF injection or implantation can decrease the expression of pro-inflammatory cytokines tumour necrosis factor α and interleukin (IL)-6, as well as lower the serum level of tumour necrosis factor γ and IL-12[8,11]. The reduction of inflammatory cytokines can reduce inflammation response, thus creating an environment capable of tissue regeneration. Finally, ASCs, as an important component of SVF, have also been shown to have immunomodulatory effects. These effects are mediated by cell-cell contact and secretory molecules. For example, ASCs can inhibit the proliferative activity of T cells partially by programmed cell death protein-1’s ligation. Then, cytokine secretion will be induced, leading to the modulation of immune cells. For instance, human leukocyte antigen-G5 secretion can be promoted, which inhibits the proliferative activity of T cells[12]. Other factors such as prostaglandin E2 and hepatic growth factor can inhibit dendritic cell function, decrease natural killer cell cytotoxicity, and promote T regulatory expansion[13].

Proangiogenic effect

Angiogenesis is the process of the generation of new vessels. It can be broadly classified into two types based on mechanism: Sprouting angiogenesis and intussusceptive angiogenesis. In sprouting angiogenesis, the endothelial cells are activated by angiogenic factors and cytokines in the microenvironment that can be secreted by macrophages[14,15]. After activation, endothelial cells degrade the basement membrane via matrix metalloproteinase and initiate sprouting. Then, they can differentiate into tip endothelial cells and stalk endothelial cells, with tip cells guiding migration and stalk cells proliferating to elongate and sprout. Pericytes are then recruited for stabilization of the blood vessel. In intussusceptive angiogenesis, new blood vessels derive from the splitting of pre-existing vessels. The key step of this process is the formation of the transluminal bridge, where endothelial cells meet to form interstitial pillars. Subsequently, the myofibroblasts and pericytes will migrate to the site of these pillars, facilitating the growth of supporting tissues[16].

The angiogenesis effect of SVF can be understood as the co-function of its cellular compositions, including ASC, endothelial progenitor cells that mature into endothelial cells, pericytes, and immune cells such as macrophages. Regarding ASC, it has been extensively reviewed by Krawczenko and Klimczak[17]. Thus, in this section, we will focus on endothelial progenitor cells, pericytes, and macrophages to gain deeper insights into how SVF facilitates angiogenesis.

Endothelial progenitor cells: Endothelial progenitor cells play a pivotal role in the angiogenesis process. They are primarily located in bone marrow but can respond to proangiogenic factors and injury signals to migrate into the bloodstream and be transported to the site for angiogenesis. Ten to twenty days are needed for the maturation of endothelial progenitor cells and this process is mediated by a variety of molecules including vascular endothelial growth factor (VEGF)-1, fibroblast growth factor 2, and collagens[18]. Endothelial progenitor cells have the ability to differentiate into endothelial cells that can be incorporated into newly forming vessels. In the meantime, they also secrete pro-angiogenic factors such as VEGF, stromal cell-derived factor-1, and insulin-like growth factor 1, which stimulate endothelial cells to migrate and proliferate and recruit tissue-resident progenitor cells[19]. Apart from pro-angiogenic factors, research has also shown that exosomes packed with microRNA derived from endothelial progenitor cells play a role in driving vascular growth and regeneration, especially under hypoxia conditions[20]. Additionally, endothelial progenitor cells have been applied to surgical replacement in vascular grafts for cardiovascular disease for their ability to differentiate into endothelial cells[21]. The tip cells are the selected endothelial cells under the activation of Notch signaling, which is mediated by the binding of ligands such as Dll4 and Jagged 1 with Notch receptors 1 to 4. The tip cells will then extend under the guidance of the spatial gradient of VEGFA[22].

Pericytes: Pericytes, another essential cell type in SVF, are usually distributed along vessels and the phenotypes vary in different types of vessels. They are concluded to be abundant in immature angiogenic sprouts and regulate various stages of angiogenesis, including vessel sprouting, maturation, and stabilization. Additionally, they regulate endothelial cell behavior by secreting cellular factors including VEGF, platelet-derived growth factor, and transforming growth factor β, and they also have the receptors to respond to angiogenic signals in the microenvironment[23,24]. Additionally, pericytes are also engaged in molecular signaling. Pericytes can modulate Rho GTPase signaling to maintain vascular stability, and they also influence endothelial cells via Tie2 receptors. Angiopoietin-2 secreted by pericytes binds to Tie2 on endothelial cells and transduces signals allowing the endothelial cells’ survival, blood vessel stabilization, and maturation[25]. Pericytes can also regulate endothelial cell behavior by controlling basement membrane degradation through matrix metalloproteinases and cathepsins, allowing vessel remodeling[26,27]. Lastly, pericytes promote the deposition of extracellular matrix (ECM) components that strengthen blood vessel walls, stabilizing blood vessels[28].

Macrophages: Macrophages are also contained in SVF and participate in multiple functions including angiogenesis. They are an important source of VEGFA that promotes new blood vessel formation[29]. It was also supposed that macrophage infiltration peaks can be found during angiogenesis since they can phagocytize cells for the ingrowth of new vessels[12]. The infiltration of macrophages in tissue is associated with their matrix metalloproteinase secretion that remodels ECM, which also creates space for new vessel growth[30]. Macrophages also secrete direction-guiding molecules such as semaphorins to guide the growth of sprouts[31]. Moreover, the shift between the M1 and M2 states of macrophages allows the dynamic regulation of angiogenesis in tissue repair. Normally, a proportion of macrophages can be induced into pro-inflammatory M1 macrophages due to the IL-4 and IL-13 in the microenvironment, while some macrophages are alternatively activated as anti-inflammatory M2 macrophages that also participate in tissue repair and wound healing. M2 macrophages can secrete anti-inflammatory cytokines and growth factors, thus promoting the formation of new blood vessels by improving the recruitment and proliferation of endothelial cells. M2 macrophages can secrete platelet-derived growth factor B that helps recruit pericytes and promote their differentiation[12,32]. M2 macrophages can release exosomes as well. These exosomes carry proangiogenic molecules and are loaded with non-coding RNAs such as miR-132-3p, which can deactivate the expression of a pro-angiogenic gene - THBS1 in endothelial cells[33].

Tissue repair and regeneration

Tissue repair and regeneration is an integrated process that is associated with angiogenesis and immunomodulation. Vascularisation enhances tissue repair, and immunomodulatory effects contribute to the healing of inflammation, which is crucial for conditions such as chronic wounds. ASC in SVF is critical for tissue repair and regeneration. Regarding the specific mechanism, the widely accepted hypothesis is that ASCs act via paracrine signaling. They secrete soluble trophic factors such as growth factors to attract tissue-specific progenitor cells to migrate and differentiate at the injury site[34]. Another hypothesis is that ASCs themselves differentiate into the cells needed for repair directly, once they are engrafted into the site of tissue damage[35,36]. In addition to ASC’s function, the fibroblasts in SVF can promote tissue repair and regeneration by modulating the remodeling of ECM. For example, they can secrete matrix metalloproteinases to allow cells to migrate to the site with injury and proliferate[37,38]. Regions administered with SVF have high levels of ECM proteins such as fibronectin and elastin that contribute to the scaffold of tissue[38]. Furthermore, fibroblasts within the SVF regulate the composition of the cytoskeleton in ECM[39]. For example, fibroblasts can secrete collagen, the most abundant molecule in the ECM and an essential component of the cytoskeleton, contributing to the integrity of repaired tissue. Meanwhile, collagen participates in regulating cell growth. For instance, collagen can inhibit EGF signaling via a protein kinase A-dependent pathway, preventing excessive growth and proliferation of liver cells. Other proteins including functional proteins can also be alternated[40,41].

APPLICATION OF SVF IN REPRODUCTIVE DISORDERS

The application of SVF in reproductive disorders has a promising future to improve infertility, which is a globally concerning problem. Infertility is defined as the inability of a couple to become pregnant within one year of regular intercourse without protection[42]. According to the analysis of data from 101 countries and 277 health surveys, the infertility trend in the 20-year period between 1990 and 2010 remains increased. People in economically developed areas are more inclined to primary infertility and secondary infertility is more prevalent in less developed areas[43].

Male reproductive disorders

In the discussion of male reproductive disorders, the caused infertility mostly comes from defective sperm quantity and quality, as well as the ejection of sperm during intercourse. Other causes include psychological factors, hormone dysregulation, genetic defects, and lifestyle, such as smoking and a sedentary lifestyle[44]. This section will focus on the impact of ASC on male reproductive disorders that are representative of the most common mechanisms, which include erectile dysfunction (ED) and testis injury.

ED: ED is the reduced ability of the penis to function for sex. Due to the complexity of the function of the penis, ED can arise from various factors. The most common factors include psychological conditions, aging, diabetes, and central nervous injury[45]. Phosphodiesterase type 5 inhibitors are the first-line treatment of ED currently and it works by improving the effect of nitric oxide to increase the blood flow for erection. However, not all patients are sensitive to phosphodiesterase type 5 inhibitors and other therapies such as hormone therapy or surgery may be applied. Using SVF is one future direction of emerging therapies for ED[45,46].

Many studies have shown that SVF therapy can improve erectile function in animals with ED. Ryu et al[47] and Song et al[48] suggest that SVF single injection in mice with diabetes-induced ED leads to improvement in erectile function reaching up to 82% of control values. Improved cavernous endothelial regeneration, increased endothelial nitric oxide synthase phosphorylation, and VEGF expression can also be observed, which indicates the promotion of angiogenesis and vascular repair[47,48]. In addition, neuroregeneration was also found to be improved by SVF therapy. It was shown that combined therapy of SVF injection and Ad-COMP-Ang1 gene therapy on diabetes-induced mice. Moreover, the reduction of fibrosis in the corpus cavernosum can also be achieved by SVF therapy[49,50]. Since fibrosis is associated with tissue remodeling and scarring, this finding might be the result of SVF’s ability to modulate the ECM. In 2016, a phase I clinical trial accessed the safety and feasibility of SVF therapy in treating ED in men[51]. The results show that SVF can be a viable future therapy for patients resistant to phosphodiesterase type 5 inhibitors with tolerable adverse events[51]. More importantly, it was highlighted that paracrine signaling might play a major role in the therapeutic effects of SVF, with secretion of VEGF that improves cavernous endothelial cell proliferation[52]. There are several challenges in SVF treatment. For instance, the preparation, optimal timing, dosing, and method for SVF delivery should be standardized to maximize therapeutic outcomes. Furthermore, many current studies in this field are not multi-center studies and only include a small number of patients. In the future, more randomized trials with a larger population of patients should be carried out. Additionally, SVF can be researched in more combinations, such as with lifestyle modification to explore the potential for diverse application of SVF.

Testicular injury: Testicular injury, which is mostly caused by trauma and torsion, often leads to male infertility and defective hormone secretion. Its current therapy mainly aims to restore the function of the testicle and alleviate symptoms, as there is only a narrow window for emergency operations to be performed to prevent irreversible injury[53]. Regenerative medicine has the potential to shed new light on testicular tissue repair and accelerate healing.

SVF has been shown to have beneficial effects in treating testicular injury. In 2019, the first study in this field, carried out by Zhou et al[54], showed that SVF has a protective effect against torsion-detorsion-induced testicular injury. The autologous SVF was observed to be integrated into testicular tissue and vessels, with the secretion of trophic factors and decreased oxidative stress. However, the production of anti-sperm antibodies should be monitored in the long term to ensure the safety of SVF injection[54]. In the following years, some studies investigated the effects of SVF treatment on testicular damage[55,56]. The results are similar, showing SVF has antioxidative effects and cell necrosis can be turned into apoptosis. Spermatozoon quantity and abnormal tail number were also found to be improved, indicating the effect of promoting sperm quality[55,56]. The transition of cell death will contribute to preserving healthy tissue and supporting the recovery of testicular injury. The current studies on SVF in treating testicular injury are not abundant. ASC also received wide attention as a potential regenerative therapy for testicular injury. For instance, Yang et al’s research showed that ASC treatment increases the trophic factor concentration in the penis in aged mice, with cavernous smooth muscle content associated with increased[57]. Additionally, innovative methods that assist in the retaining or functioning of ASC in the corpus cavernosum have been raised. Due to the rich supply of blood in the penis, the nanotechnology with the magnetic field by Wu et al[58] has been proven to improve the intracavernous pressure significantly higher than normal intracavernous injection[58]. The exploration can give us a deeper insight into the field of SVF application. Comparative studies between SVF and ASCs can be done for an improved understanding of efficacy and adverse effects. Meanwhile, in treating testicular injury, whether SVF cells themselves differentiate into Leydig cells or germs cells, or whether they mainly improve recovery by paracrine effects needs to be elucidated. Lastly, combined therapy with antioxidants or hormone therapies can be explored to maximize the efficacy.

Female reproductive disorder

On our current understanding, the research on SVF for female reproductive disorders is relatively sparse. However, in recent years, there have been emerging studies focusing on intrauterine adhesion and stress urinary incontinence treatment. Thus, we focus specifically on the potential of SVF in treating these two diseases.

Intrauterine adhesion: Intrauterine adhesion, also known as Asherman syndrome, is a condition characterized by adhesions in the uterine and endocervix and thin endometrium, of which patients often have symptoms of amenorrhea, infertility, and abortion. The conventional treatment is hysteroscopic adhesiolysis, which is often followed by supplementary therapy such as hormone replacement. However, the recurrence rate can be up to 41.9% for patients with high grades of adhesions. There is a need for new therapy to improve intrauterine adhesion[59].

SVF shows potential in treating thin endometrium, with research using animal models and clinical trials. It was shown that SVF can reverse fibrosis and increase endometrial thickness while increasing VEGF expression in Asherman’s syndrome mice[60]. Lee et al[61] also found that autologous SVF transplantation can increase endometrial thickness and improve the menstrual cycle in women with Asherman’s syndrome. However, there is also a study that shows the opposite results. Hunter et al[62] reported that SVF failed to cause a significant improvement in endometrial thickness and gland restoration. They suppose that it can be caused by insufficient SVF engraftment[62]. In 2022, a single-center cohort started to investigate the efficacy and safety of SVF treatment in patients with thin endometrium[63]. This is the first clinical trial that delves into the effectiveness of SVF treatment in patients with thin endometrium. However, the results have not been published due to the following observations and monitoring is still ongoing[63]. To sum up, there is a need for optimized delivery methods to enhance SVF effectiveness, and multi-center studies with more patients will give more confidence in assessing the SVF’s potential. Meanwhile, the precise mechanisms of how SVF improves intrauterine adhesion can be understood deeply, for the current study still cannot reveal the underlying mechanisms.

Stress urinary incontinence: Urinary continence is a condition of involuntary urine loss. Stress urinary continence (SUI) specifically refers to the involuntary urine loss caused by increased intra-abdominal pressure. Coughing, sneezing, or exhaustion are all common intriguing factors[64]. For patients with mild SUI, changing lifestyle, pelvic floor rehabilitation, and local therapy are commonly used for primary treatment. Laser therapy is often used for mild-moderate SUI. Apart from these conservative therapies, surgery can be used as well, though the surgical therapy should only be conducted after the conservative therapies have failed. However, new therapeutic strategies are still needed to solve the problems of insufficient treatment efficacy, long operation time, and surgical complications[65].

In 2018, Inoue et al[66] showed that SVF has therapeutic potential in treating SUI using mice models. The autologous SVF injection into the external urethral sphincter leads to restoration of leak point pressure and tissue repair. Meanwhile, they found the SVF cells can persist for more than four weeks after injection, which means the effect has relatively long effects[66]. Later, in 2022, Maene et al[67] conducted the first clinical trial of SVF in treating SUI. The transplantation of SVF is combined with leukocyte- and platelet-rich fibrin implantation. The results showed improvement in SUI symptoms and quality of life. Nine months of monitoring were conducted on patients, and by the end of the trial, only one patient did not have a significant improvement in urinary loss[67]. It indicates that SVF can potentially have long-term effects, which aligns with the results of Inoue et al’s research[66]. Nevertheless, there are limitations in current studies and understanding. First, the mechanism by which SVF alleviates SUI symptoms is unclear. For example, Inoue et al’s study failed to show muscle or nerve tissue regeneration which is supposed to improve sphincter function directly, though increased ECM synthesis was discovered[66]. Second, the treatment protocols of existing research vary. It complicates the comparison of results. Lastly, not all improvements in symptoms have good durability, as is shown in Maene et al’s article[67]. Repetition of treatments or other strategies of combination therapies might solve this problem. For future research, SVF preparation and delivery methods should be standardized. In addition, advanced techniques and molecular analyses can be carried out to specify the underlying precise mechanism. Moreover, large-scale trials with extended follow-up time and more patients are necessary.

CONCLUSION

In conclusion, there is emerging research showing the role of SVF in immunomodulation, angiogenesis, and tissue repair (Figure 1). SVF holds a future for the treatment of both male and female reproductive disorders due to its diverse cellular composition, regenerative nature, and source with easy access. Despite these findings, there are still challenges in the way for SVF to be widely used clinically. First, standardizing protocol is a vital direction. Since SVF consists of many cell types with its own characteristic surface markers, it is particularly important to determine a comprehensive and practical set of markers and set a uniform procedure for enzymatic preparation. Second, investigation of specific mechanisms of SVF functions under different conditions remains a problem, which should be further addressed with advanced methodologies. For example, the use of single cell RNA sequencing might help shed new light in studying SVF’s heterogeneous function, especially on cell-cell interaction in immune reaction, angiogenesis, and tissue repair. Using a 3D culture system is also a possible direction as they can mimic in vivo conditions such as hypoxia. This can potentially improve the understanding of targeted delivery of SVF cells to sites of injury for regenerative applications. Finally, to provide high-quality evidence, well-designed multi-center clinical trials should be further conducted. Except for the diseases mentioned in this article, SVF treatment for other reproductive diseases such as premature ovarian failure and polycystic ovary syndrome can also be explored in the future.

Figure 1 Summary of components, mechanisms, and application of stromal vascular fraction in reproductive therapy. Stromal vascular fraction (SVF) contains adipose-derived stem cells, pericytes, endothelial progenitor cells, and immune cells. It has an immunomodulatory function, and proangiogenic effect, and contributes to tissue repair and regeneration. For immunomodulatory functions, SVF can directly modulate both innate and adaptive immune cell functions. It can also suppress pro-inflammatory cytokines and regulate immune tolerance via cell-cell interaction and secretory molecules. For proangiogenic effect, endothelial progenitor cells, pericytes, and macrophages cooperate to secrete pro-angiogenic factors, differentiate into cells that structure new blood vessels, and contribute to vessel stabilization. For tissue repair and regeneration, SVF can either differentiate into repair cells or attract progenitor cells to differentiate into target cells via trophic factors secretion. Fibroblasts in SVF can remodel the extracellular matrix by secreting collagen and metalloproteinase. In male reproductive disorders, SVF has the potential to be applied to the treatment of erectile dysfunction and testicular injury. In female reproductive disorders, SVF has the potential to be applied to the treatment of intrauterine adhesion and stress urinary incontinence. SVF: Stromal vascular fraction; Treg: Regulatory T; IL: Interleukin; TGF: Transforming growth factor; TNF: Tumour necrosis factor; PD-1: Programmed cell death protein-1; PD-L1: Programmed death-ligand 1; HLA: Human leukocyte antigen; PGE2: Prostaglandin E2; HGF: Hepatic growth factor; NK: Natural killer; PDGF: Platelet-derived growth factor; VEGF: Vascular endothelial growth factor; ECM: Extracellular matrix; NF-κB: Nuclear factor-kappaB.