ADSC-Exs Suppresses the Fibrosis Process of Derma in Secondary Lymphedema

ADSC-Exs Suppresses the Fibrosis Process of Derma in Secondary Lymphedema | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article ADSC-Exs Suppresses the Fibrosis Process of Derma in Secondary Lymphedema Xinxin Wang, Yilan Li, Jianping Ye, Xiwen Ma, Zhenyu Wang, Xiang Guo, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5281424/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Mesenchymal stem cells (MSCs) and their exosomes, particularly adipose-derived stem cell exosomes (ADSC-Exs), have shown promise in treating secondary lymphedema (SLE), a condition characterized by fibrosis driven by the TGFβ-Smad signaling pathway. While ADSCs and ADSC-Exs have demonstrated antifibrotic effects, it is not yet clear whether these benefits stem from their ability to regulate this pathway. This study aimed to clarify the role of ADSCs and ADSC-Exs in reducing fibrosis in SLE by modulating the TGFβ-Smad pathway. Methods We established a secondary lymphedema model in C57BL/6 mice through surgical excision and localized radiation. Tissue staining was used to assess fibrosis progression at key time points, identifying the peak fibrosis stage. ADSCs and ADSC-Exs were injected into the affected areas to test their therapeutic effects, while TGFβ1 inhibitors were used as controls to block the TGFβ-Smad signaling pathway. This study compared the effects of ADSCs, ADSC-Exs, and the inhibitors on lymphedema and fibrosis markers, with a focus on their influence on the TGFβ-Smad pathway. Results Fibrosis in the SLE model peaked between the 4th and 5th weeks. Both ADSCs, ADSC-Exs, and the TGFβ inhibitor EW-7197 reduced edema and fibrosis, with ADSC-Exs having the most significant effect on skin fibrosis. This was evident by decreased levels of TGFβ1, Smad2/3, and phosphorylated Smad2/3, along with increased Smad7 levels, indicating that ADSC-Exs effectively regulate the TGFβ-Smad pathway to reduce fibrosis. Conclusions Our findings demonstrate that ADSCs and ADSC-Exs significantly alleviate edema and fibrosis in a secondary lymphedema mouse model. This therapeutic effect is largely mediated through the regulation of the TGFβ-Smad pathway, suggesting a promising approach for treating fibrosis in SLE. Adipose-derived stem cells Exosomes Lymphedema Fibrosis TGFβ-Smad pathway Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Secondary lymphedema (SLE) is a common and challenging complication for patients who undergo treatments such as surgery or radiation therapy, especially in the context of breast or pelvic cancer[ 1 , 2 ]. Globally, approximately 200 million people suffer from SLE, and with increasing cancer rates, its incidence is expected to rise[ 3 , 4 ]. Unfortunately, there is no definitive cure for SLE. While physical therapy, surgical interventions, and cytokine-based treatments offer symptomatic relief, none effectively prevent disease progression or fibrosis. The most widely accepted standard for managing SLE is comprehensive decongestive therapy, which includes manual lymphatic drainage, intermittent pneumatic compression, bandaging, and skincare[ 5 , 6 ]. Surgical options such as lymphaticovenous anastomosis and liposuction are available but do not prevent the advancement of fibrosis or infections[ 7 , 8 ]. Experimental approaches have focused on enhancing lymphatic endothelial cell (LEC) proliferation, regulating inflammation, and reducing fibrosis[ 9 ]. Mesenchymal stem cells (MSCs), known for their regenerative capabilities, have shown promise in alleviating edema and supporting LEC growth[ 10 , 11 ]. For example, studies on breast cancer patients with lymphedema demonstrated an 81% reduction in edema volume when treated with autologous bone marrow MSCs alongside decongestive therapy, compared with a 55% reduction with therapy alone[ 12 ]. Despite these promising results, MSC treatments are hindered by low survival rates and diminishing efficacy over time, leading researchers to seek more effective alternatives[ 13 , 14 ]. Adipose-derived stem cell exosomes (ADSC-Exs) offer a promising solution[ 15 ]. These nanovesicles, 40–150 nm in size, contain biologically active molecules, including lipids, cytokines, mRNAs, and microRNAs, which mediate tissue responses[ 16 – 18 ]. Compared with MSCs, ADSC-Exs present several advantages, including greater stability, a lower risk of immune rejection, and no risk of uncontrolled proliferation[ 19 ]. While research indicates that exosomes from mesenchymal stem cells or engineering exosomes can reduce edema and fibrosis in lymphedema[ 20 , 21 ], the specific mechanisms, especially those related to fibrosis control, are still unclear. Further investigation are needed to determine the full therapeutic potential of ADSC-Exs in the treatment of SLE. Addressing fibrosis in SLE patients is critical for patient outcomes, as fibrosis in these patients is largely driven by the activation of the TGFβ-Smad signaling pathway[ 22 , 23 ]. Under normal conditions, TGFβ remains inactive in the extracellular matrix, but once activated, it triggers Smad proteins, which promote fibrosis. As SLE progresses, collagen deposition in the skin increases, and TGF-β1 activation intensifies, amplifying the fibrotic response[ 24 ]. This study aimed to evaluate the role of ADSCs, ADSC-Exs, and the TGFβ1 inhibitor EW-7197 in reducing fibrosis in SLE through the modulation of the TGFβ-Smad pathway. By investigating whether ADSC-Exs can inhibit fibrosis by targeting this pathway, this study provides a novel therapeutic approach for managing fibrosis in SLE patients. Methods ADSC and ADSC-Ex isolation and identification Adipose derived mesenchymal stem cells (ADSCs) were collected from adipose tissue isolated from patients who underwent liposuction at Zhengzhou Central Hospital Affiliated Zhengzhou University. This project was approved by the Medical Ethics Committee of Zhengzhou Central Hospital (Approval number: 202291. Date of approval: January 6, 2022). ADSCs were identified via functional assays including adipogenic and osteogenic differentiation, as well as flow cytometry to detect surface proteins specific to ADSCs. The extracted ADSCs expressed CD73, CD90 and CD105, but did not express CD34 or CD45[ 25 ]. Adipose derived mesenchymal stem cells exosomes (ADSC-Exs) were collected from ADSCs via ultracentrifugation. A BCA protein assay was used to determine the protein concentration, and nanoparticle tracking analysis and Western blot analysis were used to assess the characteristics and transmembrane proteins CD9, CD81 and CD63 [ 26 ]. The corresponding nanoparticle analysis is included as Figure S1 in the Supplementary Material. Cell viability assay A total of 8🞨10 3 LECs were plated in 96-well plates and cultured in 100 µL of ECM medium overnight. After 24 hours, the medium from the wells was discarded, and PBS or exosomes(50µg) were added for culture. Three replicates were used for for each group. CCK-8 assays were performed after 24 and 48 hours to measure cell viability[ 27 ]. Transwell migration and invasion assay The cells were plated in a 6-well plate, with three replicates per group. Once the cells had adhered, they were treated according to the experimental groups. After 24 hours, the cells were digested and collected, after which the cells were counted after resuspension. Next, a 200 µl cell suspension (approximately 70,000 cells) was prepared. In a 24-well plate, 700 µl of complete medium was added to each well, and a chamber containing 200 µl of serum-free cell suspension was placed into each well. After incubation for 24 hours, the chamber was washed three times with 200 µl of PBS, and then, the cells inside the chamber were gently scraped off with a cotton swab dipped in PBS. The cells were fixed by placing the chamber in pure methanol (700 µl in the bottom of the well, 200 µl inside the chamber) for 20 minutes at room temperature. The methanol was washed away slowly with PBS. The cells were incubated at room temperature in the dark for 1–2 hours, after which they were rinsed with distilled water containing crystal violet dye. The chamber was then placed in a 24-well plate, filled with water, and observed under a microscope (10x magnification), at least three fields of view per well were selected for imaging[ 28 ]. ImageJ software was used for counting the cells. Transwell invasion assay followed the same procedure after Matrigel was applied to the wells. Tube formation assay One day before the experiment, the necessary tools, such as pipettes, tips, and 96-well cell culture plates, were placed into a -80°C freezer to cool them. The Matrigel was placed in a 4°C refrigerator overnight to thaw. Using cooled pipette tips, transfer 50 µL of Matrigel was transferred to each well of a 96-well plate, and spread evenly. The plate was then placed in a 37°C incubator for at least 1 hour to allow the Matrigel to solidify. The cells were resuspended at a concentration of approximately 2.0×10⁴-3.0×10⁴ cells per well in 100 µL of culture medium. After mixing well, the cell suspension was added evenly over the solidified Matrigel[ 29 ]. The samples were incubated at 37°C for 24 hours, imaging equipment was used to capture bright-field images of tube formation, and the images were analyzed via ImageJ software. SLE mouse model and experiment design We selected 67 C57BL/6 female mice, aged between 15–20 weeks to research. According to the published literatures, 27 mice were divided into 9 groups with the random number table method. We detected the fibrosis levels after SLE mouse model were constructed successfully during different time points via Masson staining assay. Then, 40 C57BL/6 female mice were divided into 5 groups with the random number table method: Sham group, SLE group, ADSC group, Ex group and EW group. Except from Sham group, others were constructed as secondary lymphedema animal models. (Ethical approval number: 202291, Date of approval: January 6, 2022) The model was constructed from the previously published literature, and the popliteal fossa was cut to remove the popliteal lymph node and the lymph node surrounding fat tissue, supplemented by 30 Gy X-ray radiation of the inguinal lymph nodes 1 week before and after surgery, to maximize the degree of clinical imitative ability of the disease[ 30 – 32 ]. The work has been reported in line with the ARRIVE guidelines 2.0. ADSC, ADSC-Exs local injection One week after SLE induction, the SLE mice were carefully grabbed and injected subcutaneously with the left hindlimb lesion (1🞨10 6 ) in phosphate buffer; the ADSC-Exs mice were injected with 140 µg of ADSC-Ex, the other mice were injected with phosphate buffer, and all the groups were injected with approximately 300 µL. Attention should be given to the depth of injection control, leakage caused by artificial efficacy error should be prevented. EW-7197 Oral Administration Using 20g of mouse as the standard, a working solution of 2mg/mL EW-7197 in DMSO was prepared according to the manufacturer's instructions. After the EW group of mice were established, they were immediately orally administered EW-7197 DMSO solution at a dosage of 20 mg/kg for two weeks, accompanied by 0.1mL of artificial gastric juice to protect the gastric mucosa[ 33 ]. Measurement of diameter, circumference and volume Hair was removed from the mice under anesthesia, and the diameters and circumferences at 2 mm above the knee were measured via a caliper and a nonelastic micro tape with three repeated measurements for an average. The volumes of edematous hind limbs were subsequently measured with water replacement, each measurement was repeated three times, and the average value was taken. All of measurement was assessed by one person, recorded by another partner. Masson Staining Analysis After tissue fixation, the samples were dehydrated and embedded in paraffin, and 4 µm-thick sections were prepared. The sections were dewaxed in water following standard procedures. Staining was performed according to the Masson staining kit protocol, followed by two rounds of dehydration with anhydrous ethanol, two rounds of clearing with xylene, and sealing with neutral resin. After drying, images were captured under a microscope, and image analysis was performed via ImageJ software. Sirius red staining analysis Following the preparation of 4 µm-thick sections, the sections were dewaxed with water. Staining was performed according to the Sirius Red staining kit protocol, followed by dehydration with anhydrous ethanol and clearing with xylene. After the samples were sealed with neutral resin and dried, images were captured via both a regular optical microscope and a polarized light microscope, and analyzed with ImageJ software. Immunohistochemical analysis After preparing 4 µm-thick sections, the sections were dewaxed in water. Antigen retrieval and blocking were performed according to standard immunohistochemical procedures. The antibody incubation solution (LYVE-1) was prepared at a 1:200 ratio and evenly applied to the sections, which were then placed in a humidified chamber and incubated overnight at 4°C. The sections were washed with PBS three times, and residual moisture was removed by shaking off the PBS. The appropriate secondary antibody (HRP-labeled) corresponding to the primary antibody species was added to cover the tissue, followed by incubation at room temperature for 50 minutes. After DAB staining and counterstaining of the nuclei, the sections were dehydrated and sealed. Images were captured under a regular optical microscope, and analyzed via ImageJ software. Western blot analysis The mice were anesthetized and fixed on a dissection board, and euthanized via the CO 2 method. Fresh tissue was collected from the edematous area of the left hind limb, and protein samples of standard concentration were extracted. After gel electrophoresis, transfer, and blocking, the samples were incubated with the appropriate primary antibodies (TGFβ1, Smad2/3, p-Smad2/3, Smad7, LYVE-1) overnight. The samples were then incubated with the internal control (β-actin), followed by exposure. Statistical analysis The statistical significance of all experiments was calculated via unpaired t-tests, one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons tests, or two-way ANOVA with Tukey’s multiple comparisons tests. The tests used are indicated in each figure legend. All the statistical analyses were performed using GraphPad Prism 9. P values < 0.05 were considered statistically significant. Significance levels are: *P < 0.05; **P < 0.01; ***P < 0.001. Results ADSC-Ex promote LEC proliferation, migration and tube formation. In vitro, we investigated whether ADCS-Ex could stimulate lymphangiogenesis by regulating lymphatic cell proliferation, migration and tube formation. The CCK-8 results revealed a significant increase in exponential cell proliferation from at 24 hours and 48 hours after ADSC-Ex treatment, compared with that in the PBS group (Fig. 1 B). A Transwell migration assay also showed that ADSC-Exs markedly increased the migration of LECs (Fig. 1 A, 1 C). LECs treated with ADSC-Exs formed more tubes than the controls did (control/PBS,) (Fig. 1 A-D). Next, we constructed secondary lymphedema animal models with C57BL/6 mice and compared the protein expression levels of the lymphatic vessel marker LYEV-1 with those in the sham group and the wild type mice group. LYVE-1 expression was greatly increased after treatment with ADSC-Exs compared with that in the other groups (Fig. 1 E, 1 F). These results showed that ADSC-Exs have positive effects on lymphatic endothelial cell proliferation, migration, and tube formation in vitro. This provides a theoretical basis for further improving related experiments. The peak of fibrosis in SLE patients occurs approximately 1 month after model completion. Determining the timing of fibrosis onset in secondary lymphedema is crucial for understanding the process of fibrosis itself. In this study, C57BL/6 mice underwent a one-week acclimation period before the lymphedema induction phase started (Fig. 2 A). The model was established via two rounds of X-ray irradiation and one surgical procedure. Specifically, the left inguinal region of the mice was subjected to 30 Gy of X-ray radiation[ 34 ], which was administered twice—once a week before and once a week after the surgery. During surgery, under anesthesia, 0.04 ml of 0.5% methylene blue solution was injected subcutaneously into the midline of the left hind paw to visualize the lymphatic vessels. After waiting for 10 minutes, the left popliteal lymph nodes and surrounding adipose tissue were excised, followed by skin closure and the application of erythromycin to prevent infection (Fig. 2 C). In view of the diverse methods used to constructed an SLE animal model and the lack of unified standards for successful construction, the criteria for successful establishment of a mouse model were formulated on the basis of patient evaluation criteria of clinical secondary lymphedema. In this study, an animal model of SLE was successfully established as follows: when the swelling side was measured at any point, the total arm circumference of the affected side was 5% larger than that of the healthy side, or the volume of the swelling side was 10–20% larger than that of the healthy side (Fig. 2 B). The significant swelling in the left hind limb of the mice confirmed successful model establishment (Fig. 2 D). To explore the progression of fibrosis in the lymphedema mouse model, Masson staining was performed at regular intervals to assess local fibrosis in the edematous regions. When surgery was used as the reference point, fibrosis peaked between weeks 4 and 5 postoperation. Over time, the level of fibrosis decreased (Fig. 2 E). This study provides valuable insight into the temporal dynamics of fibrosis in secondary lymphedema, offering a foundation for future fibrosis studies. ADSC-Exs significantly reduced edema in SLE mice. Previous studies have demonstrated that adipose-derived stem cells (ADSCs), owing to their multipotent differentiation abilities, can promote the proliferation of lymphatic endothelial cells, effectively alleviating edema in systemic lupus erythematosus (SLE) mouse models[ 35 ]. However, the potential risk of tumorigenesis associated with ADSCs at various dosages has raised concerns. ADSC-Exs, a biologically active, noncellular small molecule derived from ADSCs, has been less studied for its effects on SLE-induced edema. Research has suggested that EW-7197 can effectively inhibit edema progression and suppress the process of fibrosis. In this study, C57BL/6 mice were divided into five groups: the sham-operated group (Sham), model control group (Ctr), ADSC group, ADSC-derived exosome group (ADSC-Exs), and EW-7197 treatment group (EW). Lymphedema modeling was performed on all the groups except the Sham group. One week after successful model establishment, each group received different interventions (Fig. 3 A). The EW group was given intragastric administration of EW-7197 at a dose of 20 µg/g for two weeks, whereas the other groups received artificial gastric fluid for the same period. In the fifth week postmodeling, the diameter of the edematous limbs in each group was measured via a caliper (Fig. 3 B), the circumferences were assessed via a tape measure, and the volume of the edematous limb was evaluated via water displacement (Fig. 3 C). The measurement sites for diameter and circumference were set at 2 mm above the knee of the left hind limb, and the water displacement measurement ranged from the toe tip to 2 mm above the knee. The results revealed that all the groups exhibited signs of edema at 1 week postmodeling. From the second week, the edema in the sham group subsided, whereas it persisted in the other groups. By the third week postsurgery, the ADSC and ADSC-Exs groups showed signs of reduced edema. By the fourth and fifth weeks, ADSC, ADSC-Exs, and EW-7197 all alleviated edema to varying degrees. However, compared with the ADSC and EW groups, the ADSC-Exs group presented the most significant reduction in edema (Fig. 3 D- 3 F). We also observed an increase in LYVE-1 expression in the subcutaneous tissues of mice in the ADSC and ADSC-Ex groups, which indicates proliferation of new lymphatic endothelial cells and explains the reason for the reduction of edema (Fig. 3 G). Masson staining and Sirius red staining revealed a significant reduction in the fibrotic area in the ADSC and ADSC-Exs groups. In the fifth week after the model was established, the mice were euthanized with carbon dioxide[ 36 ], and tissue samples from the edematous limbs of each group were collected for staining. Masson’s trichrome staining and Sirius red staining were performed to assess the degree of fibrosis in the local tissues affected by edema. Under a standard optical microscope, skeletal muscle fibers in the sham group displayed an orderly arrangement with minimal fibrosis. In contrast, the Ctr group, which underwent surgery and radiation therapy, exhibited extensive fibrotic tissue. The other three groups, which were subjucted to ADSC, ADSC-Exs, and EW-7197 interventions, presented significantly reduced fibrotic areas (Fig. 4 A), with no statistically significant differences among them (Figs. 4 B, 4 C). Under polarized light microscopy, red or yellow areas primarily indicate type I collagen fibers, while green areas represente type III collagen fibers, and a loose reticular pattern composed of multiple colors indicates type II collagen fibers. As shown in Figure A, the edematous limbs of lymphedema model mice were predominantly composed of type I collagen fibers. Additionally, we observed via microscopy that the Ctr group presented a disorganized and loose tissue structure, whereas the remaining groups presented a more organized and dense tissue arrangement. These findings suggest that ADSC, ADSC-Exs, and EW-7197 interventions can partially restore skeletal muscle morphology. ADSC-Exs inhibited the expression of the TGFβ -Smad pathway Studies have shown that the TGFβ-Smad pathway is a classical signaling pathway involved in the pathological process of fibrosis in secondary lymphedema. TGFβ1 serves as the initiating factor in this pathway[ 37 ]. Upon activation, Smad2/3 undergo phosphorylation, leading to increased expression of P-Smad2/3, while Smad7 plays an inhibitory role, regulating the pathway by preventing its overactivation. We aimed to investigate whether ADSC, EW-7197, and ADSC-Exs reduce fibrosis in secondary lymphedema by inhibiting the expression of the TGFβ-Smad pathway. In this study, Western blot analysis was used to assess the expression levels of key proteins involved in this pathway to determine whether these interventions have an inhibitory effect on the pathway. Compared with those in the Ctr group, the fibrosis levels in the ADSC, ADSC-Exs, and EW-7197 groups were significantly lower. Moreover, the expression levels of TGFβ1, Smad2/3, and P-Smad2/3 were markedly lower in these groups than in the Ctr group. Additionally, in the ADSC-Exs group, Smad7 expression was elevated, further inhibiting the activation of the TGFβ-Smad pathway. In contrast, no significant increase in Smad7 expression was observed in the ADSC and EW groups, with Smad7 expression in the EW group even being lower than that in the sham group. These results suggest that ADSC, ADSC-Exs, and EW-7197 can inhibit fibrosis in mice with secondary lymphedema, primarily by suppressing the expression of TGFβ1, Smad2/3, and P-Smad2/3. Additionally, ADSC-Exs further inhibited the profibrotic effects of the TGFβ-Smad pathway by promoting Smad7 expression. These findings provide a new understanding of the mechanisms by which stem cells and their exosomes alleviate fibrosis in lymphedema, offering new insights for future drug development. Discussion Secondary lymphedema (SLE) is a multifaceted complication often associated with malignant tumors. It manifests as progressive edema, fibrosis, adipose deposition, and chronic inflammation, all of which significantly impact patients' quality of life[ 38 ]. Fibrosis, in particular, presents a profound clinical challenge, leading to sensory impairments and psychological distress[ 39 ]. Given the limited therapeutic options available, the need for new strategies to address fibrosis in SLE is paramount. The development of a reliable animal model is critical for studying SLE and testing potential therapies. In this study, we introduced a novel model that combines two 30 Gy X-ray irradiations with a semicircular resection of the popliteal lymph nodes and surrounding adipose tissue. This approach minimizes surgical exposure and adheres strictly to aseptic techniques, thereby reducing the risk of infection and necrosis. By establishing this stable and reproducible model, we lay the groundwork for future studies into SLE pathophysiology, as well as drug development and potential treatments. Understanding the timeline of fibrosis development is essential for designing effective fibrosis-related experiments. Clinically, SLE is divided into four stages: Stage 0 (latent), Stage 1 (early stasis), Stage 2 (edema with mild fibrosis), and Stage 3 (severe fibrosis with complications such as papillomatosis)[ 40 ]. Despite these classifications, the precise timing of fibrosis progression remains loosely defined, particularly in animal models. This study used Masson staining to assess fibrosis severity and progression. Our results revealed that the fibrotic area increased progressively during the first four weeks postmodeling, peaking between the fourth and fifth weeks, and plateaued thereafter. Although this timeline is specific to the C57BL/6 mouse model, it provides valuable insights for studies in other species, enabling the identification of optimal intervention points in the fibrosis process. Mesenchymal stem cells (MSCs) have long been studied for their ability to increase endothelial cell proliferation, but their therapeutic application is hampered by several limitations, including potential loss of differentiation capacity and an increased risk of malignancy with prolonged passage. In contrast, exosomes derived from MSCs, including those derived from adipose-derived stem cells (ADSCs), are promising alternatives. These exosomes lack biological activity but carry genetic materials such as miRNA and cDNA, which facilitate cellular communication[ 41 ]. Owing to their low variability, high reliability, and ease of storage make them an increasingly valuable tools in disease research[ 42 ]. Previous studies demonstrated that the injection of exosomes from umbilical cord blood MSCs into SLE mice, increased lymphatic endothelial cell (LEC) proliferation and reduced edema[ 21 ]. However, fibrosis-related changes were not addressed in that study. Building on these findings, our research explored the effects of ADSC-Exs on both edema and fibrosis in SLE patients. In vitro assays revealed that ADSC-Exs effectively promoted LEC proliferation, migration, and differentiation, confirming the results of previous studies. In the animal models, both the ADSC and ADSC-Exs groups presented significant reductions in edema compared to the control group[ 43 , 44 ], suggesting their role in facilitating the proliferation of lymphatic capillaries and establishing new lymphatic pathways. Although this study did not include direct in vivo assessments of the effects of Exs on LECs, the overall reduction in edema strongly suggests the therapeutic potential of ADSC-Exs in SLE. Weekly local injections of ADSCs or ADSC-Exs after model establishment revealed that, while edema initially increased in all groups during the first three weeks, edema in the ADSC and ADSC-Exs groups began to decrease from the third week onward, in contrast to that in the model group, which remained swollen. These findings underscore the promise of ADSC-Exs in alleviating edema, although the precise mechanisms require further investigation. TGFβ1 plays a pivotal role in epithelial-mesenchymal transition and fibrosis development, making it a key target in SLE-related research[ 22 , 45 ]. While the exact mechanisms of skin fibrosis in SLE are not fully understood, previous studies have suggested that elevated levels of chymase in SLE may activate latent TGFβ, exacerbating fibrosis. Given that TGFβ1 is a central initiator of the TGFβ-Smad pathway[ 46 ], it is plausible that this pathway is critical in regulating fibrosis in SLE. To explore this, we conducted Western blot analyses to assess the expression of key proteins in the TGFβ-Smad pathway, including TGFβ1, Smad7, Smad2/3, and phosphorylated Smad2/3. We also examined the effects of EW-7197, a competitive inhibitor of activin receptor-like kinase 5 (ALK-5) and TGFβ1[ 47 ], which has been shown to inhibit the TGFβ-Smad pathway[ 48 ]. Our findings revealed that both ADSCs and ADSC-Exs, along with EW-7197, effectively inhibited the expression of TGFβ1, and Smad2/3, and phosphorylated Smad2/3, whereas ADSC-Exs uniquely promoted the expression of Smad7, a negative feedback regulator in the TGFβ-Smad pathway. These findings indicate that ADSCs, ADSC-Exs, and EW-7197 may alleviate fibrosis by modulating the TGFβ-Smad pathway. Notably, ADSC-Exs had a more pronounced effect on inhibiting this pathway, underscoring their potential as therapeutic agents in SLE. However, it is unlikely that a single signaling pathway governs the complex inflammatory environment observed in SLE. Other factors, signaling pathways, and possibly undiscovered mechanisms may also contribute to disease progression. Additionally, our study did not explore the full range of mechanisms through which ADSCs and ADSC-Exs regulate the TGFβ-Smad pathway. Therefore, further research is necessary to better understand these processes and improve treatment strategies for SLE patients. In conclusion, ADSC-derived exosomes offer significant promise in reducing both edema and fibrosis in secondary lymphedema through the modulation of the TGFβ-Smad pathway. While the exact mechanisms of action remain to be fully elucidated, this study provides a foundation for future research and highlights the therapeutic potential of ADSC-Exs in managing SLE. Conclusions This study highlights the improved establishment of a secondary lymphedema (SLE) model by combining two 30 Gy X-ray irradiations with the surgical removal of popliteal lymph nodes, compared with the use of radiation or surgery alone. Through this stable animal model, the peak fibrosis stage was identified between the 4th and 5th weeks postmodeling. For the first time, adipose-derived stem cells (ADSCs), their exosomes (ADSC-Exs), and the TGFβ inhibitor EW-7197 were evaluated together in a lymphedema mouse model to compare their antifibrotic effects. The results demonstrated that ADSC-Exs significantly reduced both edema volume and fibrosis inpatients with SLE by downregulating TGFβ1 and phosphorylated Smad2/3, while upregulating Smad7. This modulation of the TGFβ-Smad signaling pathway effectively suppressed fibrosis, highlighting the superior antifibrotic potential of ADSC-Exs in comparison with other treatments. These findings suggest that ADSC-Exs constitute a promising therapeutic approach for SLE, particularly for targeting fibrosis through precise regulation of the TGFβ-Smad pathway. The ability of ADSC-Exs to regulate key proteins involved in fibrosis represents a potential breakthrough in the development of effective treatments for SLE. Further research is warranted to explore its full clinical potential and the mechanisms underlying its therapeutic effects. Abbreviations SLE Secondary lymphedema ADSCs Adipose derived stem cells ADSC-Exs Adipose derived stem cell exosomes MSCs Mesenchymal stem/stromal cells LECs Lymphatic endothelial cells EW EW-7197 FBS Fetal bovine serum RT Room temperature Declarations Ethics approval and consent to participate The ethical approved projects entitled “ADSC-Exs Suppresses the Fibrosis Process of Derma in Secondary Lymphedema” was approved by the Medical Ethics Committee of Zhengzhou Central Hospital. Approval number: 202291. Date of approval: January 6, 2022. The studies described adhere to the Helsinki declaration. Consent for publication Not applicable. Availability of data and material The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Funding 1.The Henan Province Key Research and Development and Promotion Special Project (Science and Technology Tackling Key Problems) (222102310657); 2.Research and Curriculum Construction Project of Graduate Education Reform in School of Medical Sciences, Zhengzhou University (040012023B066); 3.Research Project on Graduate Education of Zhengzhou University (YJSJY202344); 4. The project of Graduate Science and Technology Innovation Support Plan at Xinxiang Medical University (YJSCX202146Y) Authors' contributions The authors declare that they have not use AI-generated work in this manuscript. XW: Writing–original draft, finishing the experiment, collecting articles and drawing the figures. YL: Editing. JY: Providing suggestions and editing. XM: Editing. ZW: Providing suggestions and software helping. XG: Collecting reviews. MX: Collecting reviews. JM: Collecting reviews and helping the experiment. JW: Providing suggestions and editing. Acknowledgements The authors sincerely thank Jianping Ye (Institute of Trauma and Metabolism, Zhengzhou Central Hospital Affiliated to Zhengzhou University, Tianjian Laboratory of Advanced Biomedical Sciences, Academy of Medical Sciences, Zhengzhou University) and Quanxiang Han (Department of Radiotherapy, Zhengzhou Central Hospital Affiliated to Zhengzhou University) for their professional guidance and great help. References Sleigh BC, Manna B. Lymphedema. StatPearls. Treasure Island (FL): StatPearls PublishingCopyright © 2023. StatPearls Publishing LLC.; 2023. Brown S, Dayan JH, Kataru RP, Mehrara BJ. The Vicious Circle of Stasis, Inflammation, and Fibrosis in Lymphedema. Plast Reconstr Surg. 2023;151:e330–41. Grada AA, Phillips TJ. Lymphedema: Pathophysiology and clinical manifestations. J Am Acad Dermatol. 2017;77:1009–20. Jia L, Du Y, Chu L, Zhang Z, Li F, Lyu D, Li Y, Li Y, Zhu M, Jiao H, et al. Prevalence, risk factors, and management of dementia and mild cognitive impairment in adults aged 60 years or older in China: a cross-sectional study. Lancet Public Health. 2020;5:e661–71. Thompson B, Gaitatzis K, Janse de Jonge X, Blackwell R, Koelmeyer LA. Manual lymphatic drainage treatment for lymphedema: a systematic review of the literature. J Cancer Surviv. 2021;15:244–58. Rafn BS, Bodilsen A, von Heymann A, Lindberg MJ, Byllov S, Andreasen TG, Johansen C, Christiansen P, Zachariae R. Examining the efficacy of treatments for arm lymphedema in breast cancer survivors: an overview of systematic reviews with meta-analyses. EClinicalMedicine. 2024;67:102397. Cheng G, Duan Y, Xiong Q, Liu W, Yu F, Qing L, Wu P, Gong L, Li X, Tang J. Clinical application of magnetic resonance lymphangiography in the vascularized omental lymph nodes transfer with or without lymphaticovenous anastomosis for cancer-related lower extremity lymphedema. Quant Imaging Med Surg. 2023;13:5945–57. Shah C, Asha W, Vicini F. Current Diagnostic Tools for Breast Cancer-Related Lymphedema. Curr Oncol Rep. 2023;25:151–4. Shimizu Y, Che Y, Murohara T. Therapeutic Lymphangiogenesis Is a Promising Strategy for Secondary Lymphedema. Int J Mol Sci 2023, 24. Ehyaeeghodraty V, Molavi B, Nikbakht M, Malek Mohammadi A, Mohammadi S, Ehyaeeghodraty N, Fallahi B, Mousavi SA, Vaezi M, Sefidbakht S. Effects of mobilized peripheral blood stem cells on treatment of primary lower extremity lymphedema. J Vasc Surg Venous Lymphat Disord. 2020;8:445–51. Schaverien MV, Aldrich MB. New and Emerging Treatments for Lymphedema. Semin Plast Surg. 2018;32:48–52. Mahé P, Arsac M, Chatellier S, Monnin V, Perrot N, Mailler S, Girard V, Ramjeet M, Surre J, Lacroix B, et al. Automatic identification of mixed bacterial species fingerprints in a MALDI-TOF mass-spectrum. Bioinformatics. 2014;30:1280–6. Qian H, Ding X, Zhang J, Mao F, Sun Z, Jia H, Yin L, Wang M, Zhang X, Zhang B, et al. Cancer stemness and metastatic potential of the novel tumor cell line K3: an inner mutated cell of bone marrow-derived mesenchymal stem cells. Oncotarget. 2017;8:39522–33. Sun Z, Chen J, Zhang J, Ji R, Xu W, Zhang X, Qian H. The role and mechanism of miR-374 regulating the malignant transformation of mesenchymal stem cells. Am J Transl Res. 2018;10:3224–32. Ren Y, Zhang H. Emerging role of exosomes in vascular diseases. Front Cardiovasc Med. 2023;10:1090909. Chen H, Wang L, Zeng X, Schwarz H, Nanda HS, Peng X, Zhou Y. Exosomes, a New Star for Targeted Delivery. Front Cell Dev Biol. 2021;9:751079. An Y, Lin S, Tan X, Zhu S, Nie F, Zhen Y, Gu L, Zhang C, Wang B, Wei W, et al. Exosomes from adipose-derived stem cells and application to skin wound healing. Cell Prolif. 2021;54:e12993. Shen K, Jia Y, Wang X, Zhang J, Liu K, Wang J, Cai W, Li J, Li S, Zhao M, et al. Exosomes from adipose-derived stem cells alleviate the inflammation and oxidative stress via regulating Nrf2/HO-1 axis in macrophages. Free Radic Biol Med. 2021;165:54–66. Kalluri R, LeBleu VS. The biology, function, and biomedical applications of exosomes. Science 2020, 367. Li B, Yang J, Wang R, Li J, Li X, Zhou X, Qiu S, Weng R, Wu Z, Tang C, Li P. Delivery of vascular endothelial growth factor (VEGFC) via engineered exosomes improves lymphedema. Ann Transl Med. 2020;8:1498. Ting Z, Zhi-Xin Y, You-Wen T, Fu-Ji Y, Hui S, Fei M, Wei Z, Wen-Rong X, Hui Q, Yong-Min Y. Exosomes derived from human umbilical cord Wharton's jelly mesenchymal stem cells ameliorate experimental lymphedema. Clin Transl Med. 2021;11:e384. Itoh F, Watabe T. TGF-β signaling in lymphatic vascular vessel formation and maintenance. Front Physiol. 2022;13:1081376. Sano M, Hirakawa S, Suzuki M, Sakabe JI, Ogawa M, Yamamoto S, Hiraide T, Sasaki T, Yamamoto N, Inuzuka K, et al. Potential role of transforming growth factor-beta 1/Smad signaling in secondary lymphedema after cancer surgery. Cancer Sci. 2020;111:2620–34. Brown S, Nores GDG, Sarker A, Ly C, Li C, Park HJ, Hespe GE, Gardenier J, Kuonqui K, Campbell A, et al. Topical captopril: a promising treatment for secondary lymphedema. Transl Res. 2023;257:43–53. Alasmari WA, Hosny S, Fouad H, Quthami KA, Althobiany EAM, Faruk EM. Molecular and Cellular Mechanisms Involved in Adipose-derived stem cell and their extracellular vesicles in an Experimental Model of Cardio- renal Syndrome type 3: Histological and Biochemical Study. Tissue Cell. 2022;77:101842. Yang XX, Sun C, Wang L, Guo XL. New insight into isolation, identification techniques and medical applications of exosomes. J Control Release. 2019;308:119–29. Liang Y, Tang X, Zhang X, Cao C, Yu M, Wan M. Adipose Mesenchymal Stromal Cell-Derived Exosomes Carrying MiR-122-5p Antagonize the Inhibitory Effect of Dihydrotestosterone on Hair Follicles by Targeting the TGF-β1/SMAD3 Signaling Pathway. Int J Mol Sci 2023, 24. Justus CR, Marie MA, Sanderlin EJ, Yang LV. Transwell In Vitro Cell Migration and Invasion Assays. Methods Mol Biol. 2023;2644:349–59. Wei WF, Zhou HL, Chen PY, Huang XL, Huang L, Liang LJ, Guo CH, Zhou CF, Yu L, Fan LS, Wang W. Cancer-associated fibroblast-derived PAI-1 promotes lymphatic metastasis via the induction of EndoMT in lymphatic endothelial cells. J Exp Clin Cancer Res. 2023;42:160. Hadrian R, Palmes D. Animal Models of Secondary Lymphedema: New Approaches in the Search for Therapeutic Options. Lymphat Res Biol. 2017;15:2–16. Khan N, Huayllani MT, Lu X, Boczar D, Cinotto G, Avila FR, Guliyeva G, Forte AJ. Effects of diet-induced obesity in the development of lymphedema in the animal model: A literature review. Obes Res Clin Pract. 2022;16:197–205. Morita Y, Sakata N, Kawakami R, Shimizu M, Yoshimatsu G, Wada H, Kodama S. Establishment of a Simple, Reproducible, and Long-lasting Hind Limb Animal Model of Lymphedema. Plast Reconstr Surg Glob Open. 2023;11:e5243. Yoon SH, Kim KY, Wang Z, Park JH, Bae SM, Kim SY, Song HY, Jeon JY. EW-7197, a Transforming Growth Factor-Beta Type I Receptor Kinase Inhibitor, Ameliorates Acquired Lymphedema in a Mouse Tail Model. Lymphat Res Biol. 2020;18:433–8. Hayashida K, Yoshida S, Yoshimoto H, Fujioka M, Saijo H, Migita K, Kumaya M, Akita S. Adipose-Derived Stem Cells and Vascularized Lymph Node Transfers Successfully Treat Mouse Hindlimb Secondary Lymphedema by Early Reconnection of the Lymphatic System and Lymphangiogenesis. Plast Reconstr Surg. 2017;139:639–51. Hu LR, Pan J. Adipose-derived stem cell therapy shows promising results for secondary lymphedema. World J Stem Cells. 2020;12:612–20. Stuckey JE, Makhija SD, Reimer DC, Eswaraka JR. Effects of Different Grades of Carbon Dioxide on Euthanasia of Mice (Mus musculus). J Am Assoc Lab Anim Sci. 2023;62:430–7. Ruliffson BNK, Whittington CF. Regulating Lymphatic Vasculature in Fibrosis: Understanding the Biology to Improve the Modeling. Adv Biol (Weinh) 2023:e2200158. Brix B, Sery O, Onorato A, Ure C, Roessler A, Goswami N. Biology of Lymphedema. Biology (Basel) 2021, 10. Duhon BH, Phan TT, Taylor SL, Crescenzi RL, Rutkowski JM. Current Mechanistic Understandings of Lymphedema and Lipedema: Tales of Fluid, Fat, and Fibrosis. Int J Mol Sci 2022, 23. Campos JL, Pons G, Rodriguez E, Al-Sakkaf AM, Vela FJ, Pires L, Jara MJ, Sánchez-Margallo FM, Abellán E, Masiá J. Popliteal Vascular Lymph Node Resection in the Rabbit Hindlimb for Secondary Lymphedema Induction. J Vis Exp 2022. Hong P, Yang H, Wu Y, Li K, Tang Z. The functions and clinical application potential of exosomes derived from adipose mesenchymal stem cells: a comprehensive review. Stem Cell Res Ther. 2019;10:242. Lotfy A, AboQuella NM, Wang H. Mesenchymal stromal/stem cell (MSC)-derived exosomes in clinical trials. Stem Cell Res Ther. 2023;14:66. Erratum to delivery. of vascular endothelial growth factor (VEGFC) via engineered exosomes improves lymphedema. Ann Transl Med. 2021;9:1281. Lu JH, Hsia K, Su CK, Pan YH, Ma H, Chiou SH, Lin CH. A Novel Dressing Composed of Adipose Stem Cells and Decellularized Wharton's Jelly Facilitated Wound Healing and Relieved Lymphedema by Enhancing Angiogenesis and Lymphangiogenesis in a Rat Model. J Funct Biomater 2023, 14. Baik JE, Park HJ, Kataru RP, Savetsky IL, Ly CL, Shin J, Encarnacion EM, Cavali MR, Klang MG, Riedel E, et al. TGF-β1 mediates pathologic changes of secondary lymphedema by promoting fibrosis and inflammation. Clin Transl Med. 2022;12:e758. Liao X, Ruan X, Yao P, Yang D, Wu X, Zhou X, Jing J, Wei D, Liang Y, Zhang T, et al. LncRNA-Gm9866 promotes liver fibrosis by activating TGFβ/Smad signaling via targeting Fam98b. J Transl Med. 2023;21:778. Zhao X, Yang X, Wang X, Zhao X, Zhang Y, Liu S, Anderson GJ, Kim SJ, Li Y, Nie G. Penetration Cascade of Size Switchable Nanosystem in Desmoplastic Stroma for Improved Pancreatic Cancer Therapy. ACS Nano. 2021;15:14149–61. Chen J, Feng W, Sun M, Huang W, Wang G, Chen X, Yin Y, Chen X, Zhang B, Nie Y, et al. TGF-β1-Induced SOX18 Elevation Promotes Hepatocellular Carcinoma Progression and Metastasis Through Transcriptionally Upregulating PD-L1 and CXCL12. Gastroenterology. 2024;167:264–80. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5281424","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":372768877,"identity":"1dd365ba-a97d-45ad-b17c-90dd9b3cc89d","order_by":0,"name":"Xinxin Wang","email":"","orcid":"","institution":"Zhengzhou Central Hospital Affiliated to Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Xinxin","middleName":"","lastName":"Wang","suffix":""},{"id":372768878,"identity":"122f28a5-8cbf-4809-97e3-34936cf5471e","order_by":1,"name":"Yilan Li","email":"","orcid":"","institution":"Zhengzhou Central Hospital Affiliated to Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Yilan","middleName":"","lastName":"Li","suffix":""},{"id":372768879,"identity":"82798302-f1de-40f1-9494-0fe75363fdae","order_by":2,"name":"Jianping Ye","email":"","orcid":"","institution":"Zhengzhou Central Hospital Affiliated to Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Jianping","middleName":"","lastName":"Ye","suffix":""},{"id":372768880,"identity":"7a60cc37-0525-4751-9e70-f3d9eade9f52","order_by":3,"name":"Xiwen Ma","email":"","orcid":"","institution":"Zhengzhou Central Hospital Affiliated to Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Xiwen","middleName":"","lastName":"Ma","suffix":""},{"id":372768881,"identity":"99c3bae5-8f47-4dfb-ad2e-b3618f8628f2","order_by":4,"name":"Zhenyu Wang","email":"","orcid":"","institution":"People's Hospital of Hebi","correspondingAuthor":false,"prefix":"","firstName":"Zhenyu","middleName":"","lastName":"Wang","suffix":""},{"id":372768882,"identity":"7600526f-0f61-435e-a5c7-1fcc56cc8806","order_by":5,"name":"Xiang Guo","email":"","orcid":"","institution":"Xinxiang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiang","middleName":"","lastName":"Guo","suffix":""},{"id":372768883,"identity":"ebf6d34d-f3a2-4df3-887f-1787a02dce0c","order_by":6,"name":"Mengjia Xie","email":"","orcid":"","institution":"Xinxiang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Mengjia","middleName":"","lastName":"Xie","suffix":""},{"id":372768884,"identity":"8040e0dc-b131-4ab4-aa3c-a47c3b1b7f1a","order_by":7,"name":"Jiahui Ma","email":"","orcid":"","institution":"Zhengzhou Central Hospital Affiliated to Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Jiahui","middleName":"","lastName":"Ma","suffix":""},{"id":372768885,"identity":"461dce83-0661-4292-be8c-7cb9de5d19a7","order_by":8,"name":"Jingxin Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA80lEQVRIiWNgGAWjYLACCQMgwczA+ADCTSBeC7MB8VqggE2CKC0Gx88efmFRcMduw3HeY5U//hxm4GfPMWD4uQOPljN5aRYSBs+SZzbzpd2Q4DnMINnzxoCx9wxuLWYHcswMJAwOJ/Mz85jdMJA4zGBwI8eAmbENj5bzbyBa2IBaChIMDjPYE9RyI8f4AVCLHcgWhgMJQFskCGixv/HGDBjIhxMkm3mMJRsOpPNInHlWcLAXjxbJ/hzjzxJ/DtsbnD9j+PHHH2s5/vbkjQ9+4tECBGzSwPhIbIDyeEDEAbwagNH+8QPQgQQUjYJRMApGwUgGAA/rTRVh8vNbAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-9027-7032","institution":"Zhengzhou Central Hospital Affiliated to Zhengzhou University","correspondingAuthor":true,"prefix":"","firstName":"Jingxin","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2024-10-17 09:22:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5281424/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5281424/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":69447915,"identity":"597c7517-8803-4a8e-8f14-ec92e7948944","added_by":"auto","created_at":"2024-11-20 12:17:50","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":935220,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eADSC-Exs promote LEC proliferation, migration, and tube formation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Typical images of LECs crossing Transwell chambers and tube formation in the PBS control group and ADSC-Exs group (scale bar:200 μm);\u003c/p\u003e\n\u003cp\u003eB. LEC viability was assayed via CCK-8 assay at the indicated time points in the ADSC-Ex group and the PBS group.\u003c/p\u003e\n\u003cp\u003eC. Transwell assays were used to determine the effect of ADSC-Exs on LECs invation and migration.\u003c/p\u003e\n\u003cp\u003eD. The effect of ADSC-Exs on LEC tube formation was determined by tube formation assay.\u003c/p\u003e\n\u003cp\u003eE, F. LYVE-1 expression was determined via Western blotting. *P\u0026lt;0.05, **P 0.01, ***P\u0026lt;0.001, n=3. LEC: lymphatic endothelial cell; LYVE-1: Lymphatic vessel endothelial receptor 1, WT: wild type, Ex: exosome.\u003c/p\u003e","description":"","filename":"figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5281424/v1/464dece87276fbdb8ed3e91f.jpg"},{"id":69447916,"identity":"87c9a197-cb9f-40c9-95d2-f42a08983209","added_by":"auto","created_at":"2024-11-20 12:17:50","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1325708,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFibrosis level at different time points in animal models.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Overall arrangement at different time points.\u003c/p\u003e\n\u003cp\u003eB. Diagnostic criteria for clinical patients and animal models.\u003c/p\u003e\n\u003cp\u003eC. C57BL/6 mice were used to establisheh SLE models via radiation and surgery. The first line shows that X rays irradiated the inguinal region of C57BL/6 mice. The second line shows that the methylene blue solution developed into lymphatic vessels. The third line shows the popliteal lymph node and isolation of the main lymphatic vessel.\u003c/p\u003e\n\u003cp\u003eD. lymphedema animal model was successfully established in C57BL/6 mice.\u003c/p\u003e\n\u003cp\u003eE. Typical images of skin fibrosis at weeks 1, 2, 3, 4, 5, 7, 10, 14, and 19 postmodeling (10x magnification); n=3.\u003c/p\u003e\n\u003cp\u003eF. Percentage of fibrosis-positive area of total area; n=3.\u003c/p\u003e","description":"","filename":"figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5281424/v1/4fef519f85419d96cd0ff5dd.jpg"},{"id":69449520,"identity":"c82d7606-30fc-4e69-929f-cd07e832a913","added_by":"auto","created_at":"2024-11-20 12:33:50","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1264989,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEdema levels of the different groups at the 5\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eth\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e week.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Experimental groups and respective interventions at different time points.\u003c/p\u003e\n\u003cp\u003eB. Diameter measurement with a caliper.\u003c/p\u003e\n\u003cp\u003eC. Volume measurement with water displacement.\u003c/p\u003e\n\u003cp\u003eD. Diameter statistics for the different groups at different time points. (n=8)\u003c/p\u003e\n\u003cp\u003eE. Circumference statistics for the different groups at different time points. (n=8)\u003c/p\u003e\n\u003cp\u003eF. Volume statistics for the different groups at different time points. (n=8)\u003c/p\u003e\n\u003cp\u003eG. Immunohistochemical analysis for the different groups. (n=3)\u003c/p\u003e\n\u003cp\u003e*P\u0026lt;0.05, **P 0.01, ***P\u0026lt;0.001\u003c/p\u003e","description":"","filename":"figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5281424/v1/e1e498680b822b029633fd85.jpg"},{"id":69447919,"identity":"bd7b1f7c-7f6a-4be6-8d45-64195c049841","added_by":"auto","created_at":"2024-11-20 12:17:50","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1621571,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFibrosis levels of the different groups at the 5\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eth\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e week.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Fibrosis level of the different groups, as determined by Masson and Sirus red staining. The first two lines are images under a common light microscope. The last row shows images under a polarizing microscope. (n=3).\u003c/p\u003e\n\u003cp\u003eB. Fibrosis positive area statistics of Mmasson staining. (n=3).\u003c/p\u003e\n\u003cp\u003eC. Fibrosis positive area statistics of Sirus red staining. (n=3).\u003c/p\u003e\n\u003cp\u003e*P\u0026lt;0.05, **P 0.01, ***P\u0026lt;0.001\u003c/p\u003e","description":"","filename":"figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5281424/v1/7865a67138584fe15f856416.jpg"},{"id":69448972,"identity":"088aaaa9-1008-4a32-8305-4031e34d52f7","added_by":"auto","created_at":"2024-11-20 12:25:50","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":596133,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression levels of TGFβ-Smad in the different groups.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA-E. Representative Western blot of the level of key TGF-β/smad signaling-related proteins in edematous regions,(TGFβ1, Smad2/3, P-Smad2/3 and Smad7). (n=3), *P\u0026lt;0.05, **P 0.01, ***P\u0026lt;0.001\u003c/p\u003e","description":"","filename":"figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5281424/v1/bfe9f6835e56df4eefb333b0.jpg"},{"id":69705882,"identity":"f4b32575-0845-440b-8405-dcbfc509101a","added_by":"auto","created_at":"2024-11-23 16:25:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6391871,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5281424/v1/4c4c582e-8659-4c84-bddd-f39cc60974ea.pdf"},{"id":69448974,"identity":"8aa9fd99-ec8e-4d53-81d4-50df37e8e368","added_by":"auto","created_at":"2024-11-20 12:25:50","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":344871,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-5281424/v1/0c442c53eff013c85f4c62de.docx"}],"financialInterests":"","formattedTitle":"ADSC-Exs Suppresses the Fibrosis Process of Derma in Secondary Lymphedema","fulltext":[{"header":"Background","content":"\u003cp\u003eSecondary lymphedema (SLE) is a common and challenging complication for patients who undergo treatments such as surgery or radiation therapy, especially in the context of breast or pelvic cancer[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Globally, approximately 200\u0026nbsp;million people suffer from SLE, and with increasing cancer rates, its incidence is expected to rise[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Unfortunately, there is no definitive cure for SLE. While physical therapy, surgical interventions, and cytokine-based treatments offer symptomatic relief, none effectively prevent disease progression or fibrosis. The most widely accepted standard for managing SLE is comprehensive decongestive therapy, which includes manual lymphatic drainage, intermittent pneumatic compression, bandaging, and skincare[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Surgical options such as lymphaticovenous anastomosis and liposuction are available but do not prevent the advancement of fibrosis or infections[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eExperimental approaches have focused on enhancing lymphatic endothelial cell (LEC) proliferation, regulating inflammation, and reducing fibrosis[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Mesenchymal stem cells (MSCs), known for their regenerative capabilities, have shown promise in alleviating edema and supporting LEC growth[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. For example, studies on breast cancer patients with lymphedema demonstrated an 81% reduction in edema volume when treated with autologous bone marrow MSCs alongside decongestive therapy, compared with a 55% reduction with therapy alone[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Despite these promising results, MSC treatments are hindered by low survival rates and diminishing efficacy over time, leading researchers to seek more effective alternatives[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAdipose-derived stem cell exosomes (ADSC-Exs) offer a promising solution[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. These nanovesicles, 40\u0026ndash;150 nm in size, contain biologically active molecules, including lipids, cytokines, mRNAs, and microRNAs, which mediate tissue responses[\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Compared with MSCs, ADSC-Exs present several advantages, including greater stability, a lower risk of immune rejection, and no risk of uncontrolled proliferation[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. While research indicates that exosomes from mesenchymal stem cells or engineering exosomes can reduce edema and fibrosis in lymphedema[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], the specific mechanisms, especially those related to fibrosis control, are still unclear. Further investigation are needed to determine the full therapeutic potential of ADSC-Exs in the treatment of SLE.\u003c/p\u003e \u003cp\u003eAddressing fibrosis in SLE patients is critical for patient outcomes, as fibrosis in these patients is largely driven by the activation of the TGFβ-Smad signaling pathway[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Under normal conditions, TGFβ remains inactive in the extracellular matrix, but once activated, it triggers Smad proteins, which promote fibrosis. As SLE progresses, collagen deposition in the skin increases, and TGF-β1 activation intensifies, amplifying the fibrotic response[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study aimed to evaluate the role of ADSCs, ADSC-Exs, and the TGFβ1 inhibitor EW-7197 in reducing fibrosis in SLE through the modulation of the TGFβ-Smad pathway. By investigating whether ADSC-Exs can inhibit fibrosis by targeting this pathway, this study provides a novel therapeutic approach for managing fibrosis in SLE patients.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eADSC and ADSC-Ex isolation and identification\u003c/h2\u003e \u003cp\u003eAdipose derived mesenchymal stem cells (ADSCs) were collected from adipose tissue isolated from patients who underwent liposuction at Zhengzhou Central Hospital Affiliated Zhengzhou University. This project was approved by the Medical Ethics Committee of Zhengzhou Central Hospital (Approval number: 202291. Date of approval: January 6, 2022). ADSCs were identified via functional assays including adipogenic and osteogenic differentiation, as well as flow cytometry to detect surface proteins specific to ADSCs. The extracted ADSCs expressed CD73, CD90 and CD105, but did not express CD34 or CD45[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Adipose derived mesenchymal stem cells exosomes (ADSC-Exs) were collected from ADSCs via ultracentrifugation. A BCA protein assay was used to determine the protein concentration, and nanoparticle tracking analysis and Western blot analysis were used to assess the characteristics and transmembrane proteins CD9, CD81 and CD63 [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The corresponding nanoparticle analysis is included as Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e in the Supplementary Material.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell viability assay\u003c/h3\u003e\n\u003cp\u003eA total of 8\u0026#128936;10\u003csup\u003e3\u003c/sup\u003e LECs were plated in 96-well plates and cultured in 100 \u0026micro;L of ECM medium overnight. After 24 hours, the medium from the wells was discarded, and PBS or exosomes(50\u0026micro;g) were added for culture. Three replicates were used for for each group. CCK-8 assays were performed after 24 and 48 hours to measure cell viability[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eTranswell migration and invasion assay\u003c/h3\u003e\n\u003cp\u003eThe cells were plated in a 6-well plate, with three replicates per group. Once the cells had adhered, they were treated according to the experimental groups. After 24 hours, the cells were digested and collected, after which the cells were counted after resuspension. Next, a 200 \u0026micro;l cell suspension (approximately 70,000 cells) was prepared. In a 24-well plate, 700 \u0026micro;l of complete medium was added to each well, and a chamber containing 200 \u0026micro;l of serum-free cell suspension was placed into each well. After incubation for 24 hours, the chamber was washed three times with 200 \u0026micro;l of PBS, and then, the cells inside the chamber were gently scraped off with a cotton swab dipped in PBS. The cells were fixed by placing the chamber in pure methanol (700 \u0026micro;l in the bottom of the well, 200 \u0026micro;l inside the chamber) for 20 minutes at room temperature. The methanol was washed away slowly with PBS. The cells were incubated at room temperature in the dark for 1\u0026ndash;2 hours, after which they were rinsed with distilled water containing crystal violet dye. The chamber was then placed in a 24-well plate, filled with water, and observed under a microscope (10x magnification), at least three fields of view per well were selected for imaging[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. ImageJ software was used for counting the cells. Transwell invasion assay followed the same procedure after Matrigel was applied to the wells.\u003c/p\u003e\n\u003ch3\u003eTube formation assay\u003c/h3\u003e\n\u003cp\u003eOne day before the experiment, the necessary tools, such as pipettes, tips, and 96-well cell culture plates, were placed into a -80\u0026deg;C freezer to cool them. The Matrigel was placed in a 4\u0026deg;C refrigerator overnight to thaw. Using cooled pipette tips, transfer 50 \u0026micro;L of Matrigel was transferred to each well of a 96-well plate, and spread evenly. The plate was then placed in a 37\u0026deg;C incubator for at least 1 hour to allow the Matrigel to solidify. The cells were resuspended at a concentration of approximately 2.0\u0026times;10⁴-3.0\u0026times;10⁴ cells per well in 100 \u0026micro;L of culture medium. After mixing well, the cell suspension was added evenly over the solidified Matrigel[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The samples were incubated at 37\u0026deg;C for 24 hours, imaging equipment was used to capture bright-field images of tube formation, and the images were analyzed via ImageJ software.\u003c/p\u003e\n\u003ch3\u003eSLE mouse model and experiment design\u003c/h3\u003e\n\u003cp\u003eWe selected 67 C57BL/6 female mice, aged between 15\u0026ndash;20 weeks to research. According to the published literatures, 27 mice were divided into 9 groups with the random number table method. We detected the fibrosis levels after SLE mouse model were constructed successfully during different time points via Masson staining assay. Then, 40 C57BL/6 female mice were divided into 5 groups with the random number table method: Sham group, SLE group, ADSC group, Ex group and EW group. Except from Sham group, others were constructed as secondary lymphedema animal models. (Ethical approval number: 202291, Date of approval: January 6, 2022) The model was constructed from the previously published literature, and the popliteal fossa was cut to remove the popliteal lymph node and the lymph node surrounding fat tissue, supplemented by 30 Gy X-ray radiation of the inguinal lymph nodes 1 week before and after surgery, to maximize the degree of clinical imitative ability of the disease[\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The work has been reported in line with the ARRIVE guidelines 2.0.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eADSC, ADSC-Exs local injection\u003c/h2\u003e \u003cp\u003eOne week after SLE induction, the SLE mice were carefully grabbed and injected subcutaneously with the left hindlimb lesion (1\u0026#128936;10\u003csup\u003e6\u003c/sup\u003e) in phosphate buffer; the ADSC-Exs mice were injected with 140 \u0026micro;g of ADSC-Ex, the other mice were injected with phosphate buffer, and all the groups were injected with approximately 300 \u0026micro;L. Attention should be given to the depth of injection control, leakage caused by artificial efficacy error should be prevented.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEW-7197 Oral Administration\u003c/h3\u003e\n\u003cp\u003e Using 20g of mouse as the standard, a working solution of 2mg/mL EW-7197 in DMSO was prepared according to the manufacturer's instructions. After the EW group of mice were established, they were immediately orally administered EW-7197 DMSO solution at a dosage of 20 mg/kg for two weeks, accompanied by 0.1mL of artificial gastric juice to protect the gastric mucosa[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eMeasurement of diameter, circumference and volume\u003c/h3\u003e\n\u003cp\u003eHair was removed from the mice under anesthesia, and the diameters and circumferences at 2 mm above the knee were measured via a caliper and a nonelastic micro tape with three repeated measurements for an average. The volumes of edematous hind limbs were subsequently measured with water replacement, each measurement was repeated three times, and the average value was taken. All of measurement was assessed by one person, recorded by another partner.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMasson Staining Analysis\u003c/h2\u003e \u003cp\u003eAfter tissue fixation, the samples were dehydrated and embedded in paraffin, and 4 \u0026micro;m-thick sections were prepared. The sections were dewaxed in water following standard procedures. Staining was performed according to the Masson staining kit protocol, followed by two rounds of dehydration with anhydrous ethanol, two rounds of clearing with xylene, and sealing with neutral resin. After drying, images were captured under a microscope, and image analysis was performed via ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSirius red staining analysis\u003c/h2\u003e \u003cp\u003eFollowing the preparation of 4 \u0026micro;m-thick sections, the sections were dewaxed with water. Staining was performed according to the Sirius Red staining kit protocol, followed by dehydration with anhydrous ethanol and clearing with xylene. After the samples were sealed with neutral resin and dried, images were captured via both a regular optical microscope and a polarized light microscope, and analyzed with ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemical analysis\u003c/h2\u003e \u003cp\u003eAfter preparing 4 \u0026micro;m-thick sections, the sections were dewaxed in water. Antigen retrieval and blocking were performed according to standard immunohistochemical procedures. The antibody incubation solution (LYVE-1) was prepared at a 1:200 ratio and evenly applied to the sections, which were then placed in a humidified chamber and incubated overnight at 4\u0026deg;C. The sections were washed with PBS three times, and residual moisture was removed by shaking off the PBS. The appropriate secondary antibody (HRP-labeled) corresponding to the primary antibody species was added to cover the tissue, followed by incubation at room temperature for 50 minutes. After DAB staining and counterstaining of the nuclei, the sections were dehydrated and sealed. Images were captured under a regular optical microscope, and analyzed via ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eThe mice were anesthetized and fixed on a dissection board, and euthanized via the CO\u003csub\u003e2\u003c/sub\u003e method. Fresh tissue was collected from the edematous area of the left hind limb, and protein samples of standard concentration were extracted. After gel electrophoresis, transfer, and blocking, the samples were incubated with the appropriate primary antibodies (TGFβ1, Smad2/3, p-Smad2/3, Smad7, LYVE-1) overnight. The samples were then incubated with the internal control (β-actin), followed by exposure.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe statistical significance of all experiments was calculated via unpaired t-tests, one-way analysis of variance (ANOVA) with Tukey\u0026rsquo;s multiple comparisons tests, or two-way ANOVA with Tukey\u0026rsquo;s multiple comparisons tests. The tests used are indicated in each figure legend. All the statistical analyses were performed using GraphPad Prism 9. P values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant. Significance levels are: *P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eADSC-Ex promote LEC proliferation, migration and tube formation.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn vitro, we investigated whether ADCS-Ex could stimulate lymphangiogenesis by regulating lymphatic cell proliferation, migration and tube formation. The CCK-8 results revealed a significant increase in exponential cell proliferation from at 24 hours and 48 hours after ADSC-Ex treatment, compared with that in the PBS group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). A Transwell migration assay also showed that ADSC-Exs markedly increased the migration of LECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA,\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). LECs treated with ADSC-Exs formed more tubes than the controls did (control/PBS,) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-D). Next, we constructed secondary lymphedema animal models with C57BL/6 mice and compared the protein expression levels of the lymphatic vessel marker LYEV-1 with those in the sham group and the wild type mice group. LYVE-1 expression was greatly increased after treatment with ADSC-Exs compared with that in the other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE,\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). These results showed that ADSC-Exs have positive effects on lymphatic endothelial cell proliferation, migration, and tube formation in vitro. This provides a theoretical basis for further improving related experiments.\u003c/p\u003e \u003cb\u003eThe peak of fibrosis in SLE patients occurs approximately 1 month after model completion.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eDetermining the timing of fibrosis onset in secondary lymphedema is crucial for understanding the process of fibrosis itself. In this study, C57BL/6 mice underwent a one-week acclimation period before the lymphedema induction phase started (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The model was established via two rounds of X-ray irradiation and one surgical procedure. Specifically, the left inguinal region of the mice was subjected to 30 Gy of X-ray radiation[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], which was administered twice\u0026mdash;once a week before and once a week after the surgery. During surgery, under anesthesia, 0.04 ml of 0.5% methylene blue solution was injected subcutaneously into the midline of the left hind paw to visualize the lymphatic vessels. After waiting for 10 minutes, the left popliteal lymph nodes and surrounding adipose tissue were excised, followed by skin closure and the application of erythromycin to prevent infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). In view of the diverse methods used to constructed an SLE animal model and the lack of unified standards for successful construction, the criteria for successful establishment of a mouse model were formulated on the basis of patient evaluation criteria of clinical secondary lymphedema. In this study, an animal model of SLE was successfully established as follows: when the swelling side was measured at any point, the total arm circumference of the affected side was 5% larger than that of the healthy side, or the volume of the swelling side was 10\u0026ndash;20% larger than that of the healthy side (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The significant swelling in the left hind limb of the mice confirmed successful model establishment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eTo explore the progression of fibrosis in the lymphedema mouse model, Masson staining was performed at regular intervals to assess local fibrosis in the edematous regions. When surgery was used as the reference point, fibrosis peaked between weeks 4 and 5 postoperation. Over time, the level of fibrosis decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). This study provides valuable insight into the temporal dynamics of fibrosis in secondary lymphedema, offering a foundation for future fibrosis studies.\u003c/p\u003e\u003cp\u003e \u003cb\u003eADSC-Exs significantly reduced edema in SLE mice.\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePrevious studies have demonstrated that adipose-derived stem cells (ADSCs), owing to their multipotent differentiation abilities, can promote the proliferation of lymphatic endothelial cells, effectively alleviating edema in systemic lupus erythematosus (SLE) mouse models[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. However, the potential risk of tumorigenesis associated with ADSCs at various dosages has raised concerns. ADSC-Exs, a biologically active, noncellular small molecule derived from ADSCs, has been less studied for its effects on SLE-induced edema. Research has suggested that EW-7197 can effectively inhibit edema progression and suppress the process of fibrosis.\u003c/p\u003e \u003cp\u003eIn this study, C57BL/6 mice were divided into five groups: the sham-operated group (Sham), model control group (Ctr), ADSC group, ADSC-derived exosome group (ADSC-Exs), and EW-7197 treatment group (EW). Lymphedema modeling was performed on all the groups except the Sham group. One week after successful model establishment, each group received different interventions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The EW group was given intragastric administration of EW-7197 at a dose of 20 \u0026micro;g/g for two weeks, whereas the other groups received artificial gastric fluid for the same period. In the fifth week postmodeling, the diameter of the edematous limbs in each group was measured via a caliper (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), the circumferences were assessed via a tape measure, and the volume of the edematous limb was evaluated via water displacement (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). The measurement sites for diameter and circumference were set at 2 mm above the knee of the left hind limb, and the water displacement measurement ranged from the toe tip to 2 mm above the knee.\u003c/p\u003e \u003cp\u003eThe results revealed that all the groups exhibited signs of edema at 1 week postmodeling. From the second week, the edema in the sham group subsided, whereas it persisted in the other groups. By the third week postsurgery, the ADSC and ADSC-Exs groups showed signs of reduced edema. By the fourth and fifth weeks, ADSC, ADSC-Exs, and EW-7197 all alleviated edema to varying degrees. However, compared with the ADSC and EW groups, the ADSC-Exs group presented the most significant reduction in edema (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). We also observed an increase in LYVE-1 expression in the subcutaneous tissues of mice in the ADSC and ADSC-Ex groups, which indicates proliferation of new lymphatic endothelial cells and explains the reason for the reduction of edema (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003e \u003cb\u003eMasson staining and Sirius red staining revealed a significant reduction in the fibrotic area in the ADSC and ADSC-Exs groups.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn the fifth week after the model was established, the mice were euthanized with carbon dioxide[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], and tissue samples from the edematous limbs of each group were collected for staining. Masson\u0026rsquo;s trichrome staining and Sirius red staining were performed to assess the degree of fibrosis in the local tissues affected by edema. Under a standard optical microscope, skeletal muscle fibers in the sham group displayed an orderly arrangement with minimal fibrosis. In contrast, the Ctr group, which underwent surgery and radiation therapy, exhibited extensive fibrotic tissue. The other three groups, which were subjucted to ADSC, ADSC-Exs, and EW-7197 interventions, presented significantly reduced fibrotic areas (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), with no statistically significant differences among them (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB,\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eUnder polarized light microscopy, red or yellow areas primarily indicate type I collagen fibers, while green areas represente type III collagen fibers, and a loose reticular pattern composed of multiple colors indicates type II collagen fibers. As shown in Figure A, the edematous limbs of lymphedema model mice were predominantly composed of type I collagen fibers. Additionally, we observed via microscopy that the Ctr group presented a disorganized and loose tissue structure, whereas the remaining groups presented a more organized and dense tissue arrangement. These findings suggest that ADSC, ADSC-Exs, and EW-7197 interventions can partially restore skeletal muscle morphology.\u003c/p\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003eADSC-Exs inhibited the expression of the TGFβ -Smad pathway\u003c/h2\u003e \u003cp\u003eStudies have shown that the TGFβ-Smad pathway is a classical signaling pathway involved in the pathological process of fibrosis in secondary lymphedema. TGFβ1 serves as the initiating factor in this pathway[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Upon activation, Smad2/3 undergo phosphorylation, leading to increased expression of P-Smad2/3, while Smad7 plays an inhibitory role, regulating the pathway by preventing its overactivation.\u003c/p\u003e \u003cp\u003eWe aimed to investigate whether ADSC, EW-7197, and ADSC-Exs reduce fibrosis in secondary lymphedema by inhibiting the expression of the TGFβ-Smad pathway. In this study, Western blot analysis was used to assess the expression levels of key proteins involved in this pathway to determine whether these interventions have an inhibitory effect on the pathway. Compared with those in the Ctr group, the fibrosis levels in the ADSC, ADSC-Exs, and EW-7197 groups were significantly lower. Moreover, the expression levels of TGFβ1, Smad2/3, and P-Smad2/3 were markedly lower in these groups than in the Ctr group. Additionally, in the ADSC-Exs group, Smad7 expression was elevated, further inhibiting the activation of the TGFβ-Smad pathway. In contrast, no significant increase in Smad7 expression was observed in the ADSC and EW groups, with Smad7 expression in the EW group even being lower than that in the sham group.\u003c/p\u003e \u003cp\u003eThese results suggest that ADSC, ADSC-Exs, and EW-7197 can inhibit fibrosis in mice with secondary lymphedema, primarily by suppressing the expression of TGFβ1, Smad2/3, and P-Smad2/3. Additionally, ADSC-Exs further inhibited the profibrotic effects of the TGFβ-Smad pathway by promoting Smad7 expression. These findings provide a new understanding of the mechanisms by which stem cells and their exosomes alleviate fibrosis in lymphedema, offering new insights for future drug development.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eSecondary lymphedema (SLE) is a multifaceted complication often associated with malignant tumors. It manifests as progressive edema, fibrosis, adipose deposition, and chronic inflammation, all of which significantly impact patients' quality of life[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Fibrosis, in particular, presents a profound clinical challenge, leading to sensory impairments and psychological distress[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Given the limited therapeutic options available, the need for new strategies to address fibrosis in SLE is paramount.\u003c/p\u003e \u003cp\u003eThe development of a reliable animal model is critical for studying SLE and testing potential therapies. In this study, we introduced a novel model that combines two 30 Gy X-ray irradiations with a semicircular resection of the popliteal lymph nodes and surrounding adipose tissue. This approach minimizes surgical exposure and adheres strictly to aseptic techniques, thereby reducing the risk of infection and necrosis. By establishing this stable and reproducible model, we lay the groundwork for future studies into SLE pathophysiology, as well as drug development and potential treatments.\u003c/p\u003e \u003cp\u003eUnderstanding the timeline of fibrosis development is essential for designing effective fibrosis-related experiments. Clinically, SLE is divided into four stages: Stage 0 (latent), Stage 1 (early stasis), Stage 2 (edema with mild fibrosis), and Stage 3 (severe fibrosis with complications such as papillomatosis)[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Despite these classifications, the precise timing of fibrosis progression remains loosely defined, particularly in animal models. This study used Masson staining to assess fibrosis severity and progression. Our results revealed that the fibrotic area increased progressively during the first four weeks postmodeling, peaking between the fourth and fifth weeks, and plateaued thereafter. Although this timeline is specific to the C57BL/6 mouse model, it provides valuable insights for studies in other species, enabling the identification of optimal intervention points in the fibrosis process.\u003c/p\u003e \u003cp\u003eMesenchymal stem cells (MSCs) have long been studied for their ability to increase endothelial cell proliferation, but their therapeutic application is hampered by several limitations, including potential loss of differentiation capacity and an increased risk of malignancy with prolonged passage. In contrast, exosomes derived from MSCs, including those derived from adipose-derived stem cells (ADSCs), are promising alternatives. These exosomes lack biological activity but carry genetic materials such as miRNA and cDNA, which facilitate cellular communication[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Owing to their low variability, high reliability, and ease of storage make them an increasingly valuable tools in disease research[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Previous studies demonstrated that the injection of exosomes from umbilical cord blood MSCs into SLE mice, increased lymphatic endothelial cell (LEC) proliferation and reduced edema[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. However, fibrosis-related changes were not addressed in that study.\u003c/p\u003e \u003cp\u003eBuilding on these findings, our research explored the effects of ADSC-Exs on both edema and fibrosis in SLE patients. In vitro assays revealed that ADSC-Exs effectively promoted LEC proliferation, migration, and differentiation, confirming the results of previous studies. In the animal models, both the ADSC and ADSC-Exs groups presented significant reductions in edema compared to the control group[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], suggesting their role in facilitating the proliferation of lymphatic capillaries and establishing new lymphatic pathways. Although this study did not include direct in vivo assessments of the effects of Exs on LECs, the overall reduction in edema strongly suggests the therapeutic potential of ADSC-Exs in SLE. Weekly local injections of ADSCs or ADSC-Exs after model establishment revealed that, while edema initially increased in all groups during the first three weeks, edema in the ADSC and ADSC-Exs groups began to decrease from the third week onward, in contrast to that in the model group, which remained swollen. These findings underscore the promise of ADSC-Exs in alleviating edema, although the precise mechanisms require further investigation.\u003c/p\u003e \u003cp\u003eTGFβ1 plays a pivotal role in epithelial-mesenchymal transition and fibrosis development, making it a key target in SLE-related research[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. While the exact mechanisms of skin fibrosis in SLE are not fully understood, previous studies have suggested that elevated levels of chymase in SLE may activate latent TGFβ, exacerbating fibrosis. Given that TGFβ1 is a central initiator of the TGFβ-Smad pathway[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], it is plausible that this pathway is critical in regulating fibrosis in SLE. To explore this, we conducted Western blot analyses to assess the expression of key proteins in the TGFβ-Smad pathway, including TGFβ1, Smad7, Smad2/3, and phosphorylated Smad2/3. We also examined the effects of EW-7197, a competitive inhibitor of activin receptor-like kinase 5 (ALK-5) and TGFβ1[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], which has been shown to inhibit the TGFβ-Smad pathway[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur findings revealed that both ADSCs and ADSC-Exs, along with EW-7197, effectively inhibited the expression of TGFβ1, and Smad2/3, and phosphorylated Smad2/3, whereas ADSC-Exs uniquely promoted the expression of Smad7, a negative feedback regulator in the TGFβ-Smad pathway. These findings indicate that ADSCs, ADSC-Exs, and EW-7197 may alleviate fibrosis by modulating the TGFβ-Smad pathway. Notably, ADSC-Exs had a more pronounced effect on inhibiting this pathway, underscoring their potential as therapeutic agents in SLE. However, it is unlikely that a single signaling pathway governs the complex inflammatory environment observed in SLE. Other factors, signaling pathways, and possibly undiscovered mechanisms may also contribute to disease progression. Additionally, our study did not explore the full range of mechanisms through which ADSCs and ADSC-Exs regulate the TGFβ-Smad pathway. Therefore, further research is necessary to better understand these processes and improve treatment strategies for SLE patients.\u003c/p\u003e \u003cp\u003eIn conclusion, ADSC-derived exosomes offer significant promise in reducing both edema and fibrosis in secondary lymphedema through the modulation of the TGFβ-Smad pathway. While the exact mechanisms of action remain to be fully elucidated, this study provides a foundation for future research and highlights the therapeutic potential of ADSC-Exs in managing SLE.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study highlights the improved establishment of a secondary lymphedema (SLE) model by combining two 30 Gy X-ray irradiations with the surgical removal of popliteal lymph nodes, compared with the use of radiation or surgery alone. Through this stable animal model, the peak fibrosis stage was identified between the 4th and 5th weeks postmodeling. For the first time, adipose-derived stem cells (ADSCs), their exosomes (ADSC-Exs), and the TGFβ inhibitor EW-7197 were evaluated together in a lymphedema mouse model to compare their antifibrotic effects. The results demonstrated that ADSC-Exs significantly reduced both edema volume and fibrosis inpatients with SLE by downregulating TGFβ1 and phosphorylated Smad2/3, while upregulating Smad7. This modulation of the TGFβ-Smad signaling pathway effectively suppressed fibrosis, highlighting the superior antifibrotic potential of ADSC-Exs in comparison with other treatments.\u003c/p\u003e \u003cp\u003eThese findings suggest that ADSC-Exs constitute a promising therapeutic approach for SLE, particularly for targeting fibrosis through precise regulation of the TGFβ-Smad pathway. The ability of ADSC-Exs to regulate key proteins involved in fibrosis represents a potential breakthrough in the development of effective treatments for SLE. Further research is warranted to explore its full clinical potential and the mechanisms underlying its therapeutic effects.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eSLE Secondary lymphedema\u003c/p\u003e\u003cp\u003eADSCs Adipose derived stem cells\u003c/p\u003e\u003cp\u003eADSC-Exs Adipose derived stem cell exosomes\u003c/p\u003e\u003cp\u003eMSCs Mesenchymal stem/stromal cells\u003c/p\u003e\u003cp\u003eLECs Lymphatic endothelial cells\u003c/p\u003e\u003cp\u003eEW EW-7197\u003c/p\u003e\u003cp\u003eFBS Fetal bovine serum\u003c/p\u003e\u003cp\u003eRT Room temperature\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ethical approved projects entitled \u0026ldquo;ADSC-Exs Suppresses the Fibrosis Process of Derma in Secondary Lymphedema\u0026rdquo; was approved by the Medical Ethics Committee of\u0026nbsp;Zhengzhou Central Hospital.\u0026nbsp;Approval number: 202291. Date of approval: January 6, 2022. The studies described adhere to the Helsinki declaration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e1.The Henan Province Key Research and Development and Promotion Special Project (Science and Technology Tackling Key Problems) (222102310657);\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2.Research and Curriculum Construction Project of Graduate Education Reform in School of Medical Sciences, Zhengzhou University (040012023B066);\u003c/p\u003e\n\u003cp\u003e3.Research Project on Graduate Education of Zhengzhou University (YJSJY202344);\u003c/p\u003e\n\u003cp\u003e4. The project of Graduate Science and Technology Innovation Support Plan at Xinxiang Medical University (YJSCX202146Y)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have not use AI-generated work in this manuscript. XW: Writing\u0026ndash;original draft, finishing the experiment, collecting articles and drawing the figures. YL: Editing. JY: Providing suggestions and editing. XM: Editing. ZW: Providing suggestions and software helping. XG: Collecting reviews. MX: Collecting reviews. JM: Collecting reviews and helping the experiment. JW: Providing suggestions and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors sincerely thank Jianping Ye (Institute of Trauma and Metabolism, Zhengzhou Central Hospital Affiliated to Zhengzhou University, Tianjian Laboratory of Advanced Biomedical Sciences, Academy of Medical Sciences, Zhengzhou University) and Quanxiang Han (Department of Radiotherapy, Zhengzhou Central Hospital Affiliated to Zhengzhou University) for their professional guidance and great help.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSleigh BC, Manna B. Lymphedema. \u003cem\u003eStatPearls.\u003c/em\u003e Treasure Island (FL): StatPearls PublishingCopyright \u0026copy; 2023. StatPearls Publishing LLC.; 2023.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrown S, Dayan JH, Kataru RP, Mehrara BJ. The Vicious Circle of Stasis, Inflammation, and Fibrosis in Lymphedema. Plast Reconstr Surg. 2023;151:e330\u0026ndash;41.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGrada AA, Phillips TJ. Lymphedema: Pathophysiology and clinical manifestations. J Am Acad Dermatol. 2017;77:1009\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJia L, Du Y, Chu L, Zhang Z, Li F, Lyu D, Li Y, Li Y, Zhu M, Jiao H, et al. Prevalence, risk factors, and management of dementia and mild cognitive impairment in adults aged 60 years or older in China: a cross-sectional study. Lancet Public Health. 2020;5:e661\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThompson B, Gaitatzis K, Janse de Jonge X, Blackwell R, Koelmeyer LA. Manual lymphatic drainage treatment for lymphedema: a systematic review of the literature. J Cancer Surviv. 2021;15:244\u0026ndash;58.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRafn BS, Bodilsen A, von Heymann A, Lindberg MJ, Byllov S, Andreasen TG, Johansen C, Christiansen P, Zachariae R. Examining the efficacy of treatments for arm lymphedema in breast cancer survivors: an overview of systematic reviews with meta-analyses. EClinicalMedicine. 2024;67:102397.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheng G, Duan Y, Xiong Q, Liu W, Yu F, Qing L, Wu P, Gong L, Li X, Tang J. Clinical application of magnetic resonance lymphangiography in the vascularized omental lymph nodes transfer with or without lymphaticovenous anastomosis for cancer-related lower extremity lymphedema. Quant Imaging Med Surg. 2023;13:5945\u0026ndash;57.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShah C, Asha W, Vicini F. Current Diagnostic Tools for Breast Cancer-Related Lymphedema. Curr Oncol Rep. 2023;25:151\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShimizu Y, Che Y, Murohara T. Therapeutic Lymphangiogenesis Is a Promising Strategy for Secondary Lymphedema. Int J Mol Sci 2023, 24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEhyaeeghodraty V, Molavi B, Nikbakht M, Malek Mohammadi A, Mohammadi S, Ehyaeeghodraty N, Fallahi B, Mousavi SA, Vaezi M, Sefidbakht S. Effects of mobilized peripheral blood stem cells on treatment of primary lower extremity lymphedema. J Vasc Surg Venous Lymphat Disord. 2020;8:445\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchaverien MV, Aldrich MB. New and Emerging Treatments for Lymphedema. Semin Plast Surg. 2018;32:48\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMah\u0026eacute; P, Arsac M, Chatellier S, Monnin V, Perrot N, Mailler S, Girard V, Ramjeet M, Surre J, Lacroix B, et al. Automatic identification of mixed bacterial species fingerprints in a MALDI-TOF mass-spectrum. Bioinformatics. 2014;30:1280\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQian H, Ding X, Zhang J, Mao F, Sun Z, Jia H, Yin L, Wang M, Zhang X, Zhang B, et al. Cancer stemness and metastatic potential of the novel tumor cell line K3: an inner mutated cell of bone marrow-derived mesenchymal stem cells. Oncotarget. 2017;8:39522\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun Z, Chen J, Zhang J, Ji R, Xu W, Zhang X, Qian H. The role and mechanism of miR-374 regulating the malignant transformation of mesenchymal stem cells. Am J Transl Res. 2018;10:3224\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRen Y, Zhang H. Emerging role of exosomes in vascular diseases. Front Cardiovasc Med. 2023;10:1090909.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen H, Wang L, Zeng X, Schwarz H, Nanda HS, Peng X, Zhou Y. Exosomes, a New Star for Targeted Delivery. Front Cell Dev Biol. 2021;9:751079.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAn Y, Lin S, Tan X, Zhu S, Nie F, Zhen Y, Gu L, Zhang C, Wang B, Wei W, et al. Exosomes from adipose-derived stem cells and application to skin wound healing. Cell Prolif. 2021;54:e12993.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShen K, Jia Y, Wang X, Zhang J, Liu K, Wang J, Cai W, Li J, Li S, Zhao M, et al. Exosomes from adipose-derived stem cells alleviate the inflammation and oxidative stress via regulating Nrf2/HO-1 axis in macrophages. Free Radic Biol Med. 2021;165:54\u0026ndash;66.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKalluri R, LeBleu VS. The biology, function, and biomedical applications of exosomes. Science 2020, 367.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi B, Yang J, Wang R, Li J, Li X, Zhou X, Qiu S, Weng R, Wu Z, Tang C, Li P. Delivery of vascular endothelial growth factor (VEGFC) via engineered exosomes improves lymphedema. Ann Transl Med. 2020;8:1498.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTing Z, Zhi-Xin Y, You-Wen T, Fu-Ji Y, Hui S, Fei M, Wei Z, Wen-Rong X, Hui Q, Yong-Min Y. Exosomes derived from human umbilical cord Wharton's jelly mesenchymal stem cells ameliorate experimental lymphedema. Clin Transl Med. 2021;11:e384.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eItoh F, Watabe T. TGF-β signaling in lymphatic vascular vessel formation and maintenance. Front Physiol. 2022;13:1081376.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSano M, Hirakawa S, Suzuki M, Sakabe JI, Ogawa M, Yamamoto S, Hiraide T, Sasaki T, Yamamoto N, Inuzuka K, et al. Potential role of transforming growth factor-beta 1/Smad signaling in secondary lymphedema after cancer surgery. Cancer Sci. 2020;111:2620\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrown S, Nores GDG, Sarker A, Ly C, Li C, Park HJ, Hespe GE, Gardenier J, Kuonqui K, Campbell A, et al. Topical captopril: a promising treatment for secondary lymphedema. Transl Res. 2023;257:43\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlasmari WA, Hosny S, Fouad H, Quthami KA, Althobiany EAM, Faruk EM. Molecular and Cellular Mechanisms Involved in Adipose-derived stem cell and their extracellular vesicles in an Experimental Model of Cardio- renal Syndrome type 3: Histological and Biochemical Study. Tissue Cell. 2022;77:101842.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang XX, Sun C, Wang L, Guo XL. New insight into isolation, identification techniques and medical applications of exosomes. J Control Release. 2019;308:119\u0026ndash;29.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiang Y, Tang X, Zhang X, Cao C, Yu M, Wan M. Adipose Mesenchymal Stromal Cell-Derived Exosomes Carrying MiR-122-5p Antagonize the Inhibitory Effect of Dihydrotestosterone on Hair Follicles by Targeting the TGF-β1/SMAD3 Signaling Pathway. Int J Mol Sci 2023, 24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJustus CR, Marie MA, Sanderlin EJ, Yang LV. Transwell In Vitro Cell Migration and Invasion Assays. Methods Mol Biol. 2023;2644:349\u0026ndash;59.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWei WF, Zhou HL, Chen PY, Huang XL, Huang L, Liang LJ, Guo CH, Zhou CF, Yu L, Fan LS, Wang W. Cancer-associated fibroblast-derived PAI-1 promotes lymphatic metastasis via the induction of EndoMT in lymphatic endothelial cells. J Exp Clin Cancer Res. 2023;42:160.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHadrian R, Palmes D. Animal Models of Secondary Lymphedema: New Approaches in the Search for Therapeutic Options. Lymphat Res Biol. 2017;15:2\u0026ndash;16.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhan N, Huayllani MT, Lu X, Boczar D, Cinotto G, Avila FR, Guliyeva G, Forte AJ. Effects of diet-induced obesity in the development of lymphedema in the animal model: A literature review. Obes Res Clin Pract. 2022;16:197\u0026ndash;205.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorita Y, Sakata N, Kawakami R, Shimizu M, Yoshimatsu G, Wada H, Kodama S. Establishment of a Simple, Reproducible, and Long-lasting Hind Limb Animal Model of Lymphedema. Plast Reconstr Surg Glob Open. 2023;11:e5243.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYoon SH, Kim KY, Wang Z, Park JH, Bae SM, Kim SY, Song HY, Jeon JY. EW-7197, a Transforming Growth Factor-Beta Type I Receptor Kinase Inhibitor, Ameliorates Acquired Lymphedema in a Mouse Tail Model. Lymphat Res Biol. 2020;18:433\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHayashida K, Yoshida S, Yoshimoto H, Fujioka M, Saijo H, Migita K, Kumaya M, Akita S. Adipose-Derived Stem Cells and Vascularized Lymph Node Transfers Successfully Treat Mouse Hindlimb Secondary Lymphedema by Early Reconnection of the Lymphatic System and Lymphangiogenesis. Plast Reconstr Surg. 2017;139:639\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu LR, Pan J. Adipose-derived stem cell therapy shows promising results for secondary lymphedema. World J Stem Cells. 2020;12:612\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStuckey JE, Makhija SD, Reimer DC, Eswaraka JR. Effects of Different Grades of Carbon Dioxide on Euthanasia of Mice (Mus musculus). J Am Assoc Lab Anim Sci. 2023;62:430\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRuliffson BNK, Whittington CF. Regulating Lymphatic Vasculature in Fibrosis: Understanding the Biology to Improve the Modeling. Adv Biol (Weinh) 2023:e2200158.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrix B, Sery O, Onorato A, Ure C, Roessler A, Goswami N. Biology of Lymphedema. Biology (Basel) 2021, 10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuhon BH, Phan TT, Taylor SL, Crescenzi RL, Rutkowski JM. Current Mechanistic Understandings of Lymphedema and Lipedema: Tales of Fluid, Fat, and Fibrosis. Int J Mol Sci 2022, 23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCampos JL, Pons G, Rodriguez E, Al-Sakkaf AM, Vela FJ, Pires L, Jara MJ, S\u0026aacute;nchez-Margallo FM, Abell\u0026aacute;n E, Masi\u0026aacute; J. Popliteal Vascular Lymph Node Resection in the Rabbit Hindlimb for Secondary Lymphedema Induction. J Vis Exp 2022.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHong P, Yang H, Wu Y, Li K, Tang Z. The functions and clinical application potential of exosomes derived from adipose mesenchymal stem cells: a comprehensive review. Stem Cell Res Ther. 2019;10:242.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLotfy A, AboQuella NM, Wang H. Mesenchymal stromal/stem cell (MSC)-derived exosomes in clinical trials. Stem Cell Res Ther. 2023;14:66.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eErratum to delivery. of vascular endothelial growth factor (VEGFC) via engineered exosomes improves lymphedema. Ann Transl Med. 2021;9:1281.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu JH, Hsia K, Su CK, Pan YH, Ma H, Chiou SH, Lin CH. A Novel Dressing Composed of Adipose Stem Cells and Decellularized Wharton's Jelly Facilitated Wound Healing and Relieved Lymphedema by Enhancing Angiogenesis and Lymphangiogenesis in a Rat Model. J Funct Biomater 2023, 14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaik JE, Park HJ, Kataru RP, Savetsky IL, Ly CL, Shin J, Encarnacion EM, Cavali MR, Klang MG, Riedel E, et al. TGF-β1 mediates pathologic changes of secondary lymphedema by promoting fibrosis and inflammation. Clin Transl Med. 2022;12:e758.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiao X, Ruan X, Yao P, Yang D, Wu X, Zhou X, Jing J, Wei D, Liang Y, Zhang T, et al. LncRNA-Gm9866 promotes liver fibrosis by activating TGFβ/Smad signaling via targeting Fam98b. J Transl Med. 2023;21:778.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao X, Yang X, Wang X, Zhao X, Zhang Y, Liu S, Anderson GJ, Kim SJ, Li Y, Nie G. Penetration Cascade of Size Switchable Nanosystem in Desmoplastic Stroma for Improved Pancreatic Cancer Therapy. ACS Nano. 2021;15:14149\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen J, Feng W, Sun M, Huang W, Wang G, Chen X, Yin Y, Chen X, Zhang B, Nie Y, et al. TGF-β1-Induced SOX18 Elevation Promotes Hepatocellular Carcinoma Progression and Metastasis Through Transcriptionally Upregulating PD-L1 and CXCL12. Gastroenterology. 2024;167:264\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Adipose-derived stem cells, Exosomes, Lymphedema, Fibrosis, TGFβ-Smad pathway","lastPublishedDoi":"10.21203/rs.3.rs-5281424/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5281424/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eMesenchymal stem cells (MSCs) and their exosomes, particularly adipose-derived stem cell exosomes (ADSC-Exs), have shown promise in treating secondary lymphedema (SLE), a condition characterized by fibrosis driven by the TGFβ-Smad signaling pathway. While ADSCs and ADSC-Exs have demonstrated antifibrotic effects, it is not yet clear whether these benefits stem from their ability to regulate this pathway. This study aimed to clarify the role of ADSCs and ADSC-Exs in reducing fibrosis in SLE by modulating the TGFβ-Smad pathway.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe established a secondary lymphedema model in C57BL/6 mice through surgical excision and localized radiation. Tissue staining was used to assess fibrosis progression at key time points, identifying the peak fibrosis stage. ADSCs and ADSC-Exs were injected into the affected areas to test their therapeutic effects, while TGFβ1 inhibitors were used as controls to block the TGFβ-Smad signaling pathway. This study compared the effects of ADSCs, ADSC-Exs, and the inhibitors on lymphedema and fibrosis markers, with a focus on their influence on the TGFβ-Smad pathway.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eFibrosis in the SLE model peaked between the 4th and 5th weeks. Both ADSCs, ADSC-Exs, and the TGFβ inhibitor EW-7197 reduced edema and fibrosis, with ADSC-Exs having the most significant effect on skin fibrosis. This was evident by decreased levels of TGFβ1, Smad2/3, and phosphorylated Smad2/3, along with increased Smad7 levels, indicating that ADSC-Exs effectively regulate the TGFβ-Smad pathway to reduce fibrosis.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOur findings demonstrate that ADSCs and ADSC-Exs significantly alleviate edema and fibrosis in a secondary lymphedema mouse model. This therapeutic effect is largely mediated through the regulation of the TGFβ-Smad pathway, suggesting a promising approach for treating fibrosis in SLE.\u003c/p\u003e","manuscriptTitle":"ADSC-Exs Suppresses the Fibrosis Process of Derma in Secondary Lymphedema","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-20 12:17:45","doi":"10.21203/rs.3.rs-5281424/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8564f23e-ce6d-4f67-bb92-ca9d85505ca1","owner":[],"postedDate":"November 20th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-11-23T16:17:06+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-20 12:17:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5281424","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5281424","identity":"rs-5281424","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}