Lymphatic specification can be observed from embryonic day 9.5 (E9.5) in a subset of cells in the walls of the cardinal veins. VEGF-C and its receptor vascular endothelial growth factor receptor 3 (VEGFR3) comprise the most essential signaling pathways during initial lymphatic development, in addition to lymphatic endothelial cell (LEC) proliferation, migration, and maintenance during early embryonic growth[13-15].

Prospero homeobox protein 1 (Prox1) determines the fate of differentiation[16,17], and is a master lymphatic transcription factor during cell proliferation and the maintenance of lymphatic integrity. In Prox1 knockout mice, LEC budding is observed during the early stages of development, suggesting that the early expression of lymphatic vascular hyaluronan receptor - 1 (Lyve-1) together with that of Prox1 represents the first indication of lymphangiogenesis[18]. Coup-TFII directs the polarized expression of Prox1 in endothelial cells within the cardinal vein[19]. Notch signaling regulates normal lymphatic vessel patterning, the deletion of which leads to excessive Prox1+ LEC differentiation and lymphatic overgrowth[20]. LEC precursors migrate out of the vein under the control of the VEGF-C/VEGFR-3/Prox1 axis[21].

Mature lymphatic structures are composed of capillaries, pre-collectors, and collecting vessels, and rely on the formation of lymph sacs and lymphatic plexuses. At approximately E10.5, Prox1 + LECs begin to migrate. At E11.5, VEGF-C/VEGFR-3 signaling stimulates lymph sac morphogenesis, and its hyperactivation leads to the overgrowth of lymph sacs[22].

Lymph sacs and lymphatic plexuses undergo further remodeling to form a functional lymphatic vessel network from E15.5 to early post-birth stages[23]. The formation of lymphatic valves, the recruitment of mural granulosa cells, and the deposition of the basement membrane are signs of maturity for collecting vessels. The transition of the LEC junctions from zippers to buttons characterizes the process of lymphatic capillary maturation[24].

In animal models of lymphedema, the infiltration of lymphocytes and macrophages is observed. Lymphatic function can be improved through immunosuppressive drug targeting at T cells, including tacrolimus[31] and atorvastatin[27]. In contrast to acute inflammation, active T cells play an important role in the progressive development of lymphedema including neolymphatic vessel formation and fibrosis[4,32]. CD4+ cells play a key role in impaired lymphangiogenesis and lymphatic dysfunction, whilst the depletion of CD8+ cells has minimal effects[33]. CD4 knockout mice with acquired lymphedema exhibit lower levels of swelling and improved lymphatic function. The number of CD4+ cells is also positively associated with the severity of edema[4]. However, CD4+ T cells have different roles when cooperating with macrophages. Ogata et al[27] found that the addition of CD4+ T cells had no effect on tube formation. However, when CD4+ T cells were co-cultured with macrophages, new lymphatic tubes were observed. Macrophages are essential during lymphangiogenesis. Recent studies show that RAMP1 signaling accelerates lymphedema by inhibiting the recruitment of macrophages[34].

T cell-derived cytokines including interleukin (IL)-4, IL-13, IL-17, interferon gamma (IFN-γ), and transforming growth factor (TGF)-β1 negatively regulate lymphangiogenesis. In vitro, IL-4, IL-13, and IL-17 have been shown to inhibit LEC proliferation through the downregulation of LEC genes[35]. IL-4, IL-13, IL-17, and TGF-β1 participate in the development of fibrosis-related diseases[4]. IFN-γ and IL-17 activate macrophages through VEGF-C production during lymphangiogenesis in different disease models. IFN-γ and IL-17 can activate macrophages and enhance VEGF-C production during lymphangiogenesis of different disease models[27].

A chronic inflammatory environment initiates diverse lymphangiogenesis processes[32]. Lymphatic vessels do not reform spontaneously, and the remodeling of pre-existing vessels occurs, leading to the dilation of vessels and decreased contractile frequencies[36,37]. It remains unclear how VEGF-C/VEGFR-3 signaling regulates lymphedema-induced lymphangiogenesis.

A stable lymphatic system is critical for homeostasis, immunity, and lipid reabsorption[33]. Adipocytes are the main parenchymal cells in AT that contribute to energy storage and pathogen defense. Adipocytes are sensitive to pathological changes such as inflammation[36]. The accumulation of lymphatic fluid that contains free fatty acids can lead to fat deposition by activated adipocytes, upregulating fat differentiation genes[24,35,38]. Enhanced lipid storage leads to the hypertrophy and hyperplasia of adipocytes. In AT samples from lymphedema patients, a decrease in elastic fibers and an increase in collagen fibers are observed[30]. Active adipogenesis and fibrosis alter the physiological structure of AT.

ADSCs are multipotent cells with the potential to differentiate into multilineage cells including osteocytes, myocytes, chondrocytes, adipocytes, astrocytes, and endotheliocytes in vitro and in vivo[39]. However, significantly fewer stem cells exist in pathologic AT compared to normal AT. The differentiation potential of ADSCs to adipocytes can compensate for inadequate lipid storage capacity[40]. Lymphedema leads to the consumption of anti-inflammatory macrophages, which play an important role in the prevention of inflammation and tissue repair. Conversely, inflammatory macrophages are prevalent in hypertrophic AT[34].

Mice are the only used animal models for lymphedema, which occurs in either the hindlimbs or tails as a result of circumferential incision. Irradiation is used as an auxiliary method with circumferential incision[41,42]. ADSCs are harvested from the AT of the same species from both intraabdominal and inguinal regions. Hwang et al[43] used ADSCs isolated from the human body, and complications related to rejection reactions were not observed. VEGF-C hydrogel sheets when applied to the injection site can reduce edema 3 to 4 d post-treatment. The reduction in the circumference and volume at the edema site following the injection of ADSCs occurs within 2-4 wk of treatment.

The dose of cells injected varied from 1 × 10⁴ to 2 × 10⁶, and all showed improved lymphatic function. Yoshida et al[42] divided mice into groups injected with 1 × 10⁴, 1 × 10⁵, and 1 × 10⁶ ADSCs. The number of lymphatic vessels significantly increased at 2 wk in a dose dependent manner. Increased LYVE-1 expression with a treatment dose of 1 × 10⁶ cells was significantly higher than that with treatment doses of 1 × 10⁵ and 1 × 10⁴ cells. Likewise, a higher dose of 1 × 10⁵ showed significantly greater LYVE-1 expression than the dose of 1 × 10⁴. Stem cells were subcutaneously injected at the site of lymphedema. Shimizu et al[44] showed that ADSCs stimulate lymphangiogenesis by secreting VEGF-C and through the recruitment of lymphatic endothelial progenitor cells. Ackermann et al[45] reported that ADSC therapy promotes lymphangiogenesis and lymphedema but to lower levels than platelet-rich plasma (PRP).

Peña Quián et al[46] provided a case report on a patient with edematous lower limbs resulting from recurrent lymphangitis. Autologous ADSCs (1-2.2 × 10⁹) were injected and a greater number of new lymphatic ramifications and lymph nodes were observed at 6 mo post-treatment via lymphoscintigraphy. Toyserkani et al[47] used autologous ADSCs in a patient suffering breast cancer–related lymphedema with deformed upper limbs. Fat grafting was performed at the same time. A total of eight axilla injections were performed at a total dosage of 4.0 × 10⁷ cells. The time of follow-up was 4 mo, and positive outcomes were observed. The reduction of arm volume along with the decrease in heaviness and tension led to a lower requirement for compression therapy. In 2017, Toyserkani et al[48] enrolled ten patients to explore the feasibility and safety of ADSC therapy. Patients received the same treatment at a slightly higher dose of 5 × 10⁷ cells. However, volume reduction was not significant after 6 mo of follow-up. Up to 50% of the patients reported an alleviation of their discomfort and had a lower requirement for conservative management. In 2019, Toyserkani et al[49] performed lymphoscintigraphic evaluations after one year of follow-up. No changes in arm volume and only mild transient complications related to liposuction were noted.