The human lymphatic system is made up of lymphatic vessels, lymphatic organs, interstitial fluid, and migrating cells (Rovenská and Rovenský, 2011; Gavins et al., 2020). However, although the building blocks are diverse, the system works as a whole, and is an important part of the immune system (Rovenská and Rovenský, 2011).

The lymphatic vasculature is found in most organs and comprises lymphatic capillaries, collecting vessels with valves, and lymph nodes. The lymphatic capillaries are blind-ended and highly permeable, lined by a single layer of loosely connected endothelial cells; they merge into the collecting vessels and then into the thoracic duct and right lymphatic duct, and eventually empty lymph into the venous system (Oliver et al., 2020). Lymphatic development involves modification of a subpopulation of embryonic venous endothelial cells in the cardinal veins expressing prospero-related homeobox 1 (PROX1), a master transcription factor, into lymphatic endothelial cell (LEC) progenitors, which subsequently bud off from cardinal veins to form lymph sacs (Oliver et al., 2020). The activation of PROX1 induces chicken ovalbumin upstream promoter-transcription factor 2 (COUP-TF II) (Lee et al., 2008/09) and SRY box transcription factor 18 (SOX18) to initiate differentiation of LECs (Srinivasan et al., 2010). After forming the sacs, LECs sprout to produce peripheral lymphatic network, which is involved in regulation of the entire growth and remodeling of the lymphatic system (Escobedo and Oliver, 2017; Kim and Song, 2017; Oliver et al., 2020).

The lymphatic system is responsible for fluid transportation and maintenance of the overall fluid balance in the body. In the interstitial space, the fluid is at sub-atmospheric pressure. However, to reach the venous system it requires a pressure of approximately 20 cm H₂O. This pressure difference is overcome by active pumping of collecting lymphatic vessels. In addition, passive vessels lead to further pumping (Moore and Bertram, 2018). However, a few causes or risk factors may impair this flow system, leading to the increased volume of interstitial fluid due to either increased inflow or decreased outflow or both; thus, the fluid balance is compromised (Ridner, 2013). This emerges as swelling (edema) and is the most common clinical symptom of lymphedema (Maclellan and Greene, 2014; Azhar et al., 2020) against which potential drugs/medicines are formulated.

Lymphangiogenesis is a process in which new lymphatic vessels are generated either from pre-existing lymphatic vessels or initial formation during embryogenesis (Varner and Schwab, 2011). This is a dynamic process during embryogenesis, but is almost absent postnatal under normal physiological conditions and only occurs in pathological conditions such as tissue remodeling, inflammation, and tumor growth (Christiansen and Detmar, 2011). It was highly associated with unfavorable clinical outcomes in tumors, particularly with respect to the recurrence-free survival rate and tumor size (Sha et al., 2019).

Under physiological conditions in adults, the lymphatic network system is remodeled into mature collecting vessels owing to the strong expression of VEGF-receptor-3 (VEGFR-3) in luminal valves and less strong in lymphangion. VEGFR-3 is a cognate ligand of vascular endothelial growth factor-C (VEGF-C) (Norrmén et al., 2011). Hence, the VEGF-C)/VEGFR-3 signaling pathway is apical to lymphangiogenesis (Veikkola et al., 2001). VEGF-C is a VEGF/platelet-derived growth factor (PDGF) family homolog with cysteine-rich C-terminal. Once it is proteolytically processed, it binds to VEGFR-3, previously termed as Fms-like tyrosine kinase-4 (FLT4) and induces autophosphorylation (Joukov et al., 1996). VEGFR-3 expression is detected in LECs, whereas VEGF-C is chemostatic and expressed by immune cell infiltrate, which is primarily a macrophage subset (Gousopoulos et al., 2017). It was reported that in mouse tail lymphedema model, VEGF-C was overexpressed by CD68⁺ cells, a macrophage marker, leading to the worsening of lymphedema’s most distinctive symptom, edema, and this was positively correlated with vascular leakage that initiated the fluid influx into the tissue. Blocking VEGF-C abolished edema development and vascular leakage (Gousopoulos et al., 2017).

VEGFR-3 alone was shown to be sufficient to induce lymphangiogenesis, as suggested by Veikkola et al., who developed transgenic mice overexpressing a VEGFR-3-specific mutant of VEGF-C (VEGF-C156S) or VEGF-D in epidermal keratinocytes using the keratin 14 promoter. In this study, lymphatic vessels in the skin were observed to be induced by both transgenes without affecting the architecture of the adjacent blood vessels (Veikkola et al., 2001).

During the state of lymphedema, lymphangiogenesis is perturbed, which results in the accumulation of protein-rich interstitial fluid, and thereby swelling, in certain sites of the body. Subsequently, the fluid induces inflammatory responses, which further initiate fibrosis, adipocyte deposition, and impaired immune response and wound healing (Norrmén et al., 2011). Therefore, in the treatment of cancer, where lymphangiogenesis facilitates the spread of cancerous cells into lymph nodes, should be abrogated; therefore, anti-inflammatory drugs such as docetaxel are generally used as a therapeutic strategy. However, the use of docetaxel, being toxic, as an adjuvant chemotherapy has been reported to be associated with a high risk of lymphedema in some breast cancer cohorts (Harris et al., 2018). This is intriguing yet conflicting because lymphangiogenesis is unfavorable in cancers but favorable in lymphedema; moreover, we agreed that lymphedema itself is an adverse consequence of anticancer treatment as described earlier in this review. This strongly indicates a potential molecular crosstalk between lymphangiogenesis and tumorigenesis (Gomes et al., 2013; Lee et al., 2015), which is of major interest, particularly with regard to developing drugs against cancer without triggering lymphedema or drugs that negate the emerging clinical hallmarks of lymphedema (Anton and Rockson, 2019). As described earlier that lymphangiogenesis is initiated through the VEGF-C/VEGFR-3 signaling pathway, VEGF-C and VEGFR-3 are two main targets proposed in both synthetic-based and phytochemical-based drug platforms, which are discussed in this article (6. Phytochemical-based substances with anti-lymphedema properties).

A selective VEGFR-3 tyrosine kinase inhibitor, SAR131675, was shown to inhibit lymphangiogenesis in both in vitro and in vivo studies through regulation of the pro-inflammatory chemokine-expressing M1 macrophages, which further leads to decreased fibrosis (Hwang et al., 2019). Although the VEGF-C/VEGFR-3 axis is crucial for lymphangiogenesis to enable the trapped lymphatic fluid to re-flow properly, this might trigger a contra-productive clinical manifestation since lymphangiogenesis is supportive for cancer metastasis. This is certainly of concern for researchers and clinicians worldwide because the vast majority of patients with acquired lymphedema are receiving treatment for various stages of cancer. This urges further studies focusing not only on the crosstalk of lymphangiogenesis in simultaneous cancer and lymphedema, but also on drugs targeting other than lymphatic-related development. Anti-fibrosis and anti-adipogenesis are the two most proposed strategies, which are elucidated in the following sections.

In addition to lymphangiogenesis, fibrosis is a major hallmark of acquired lymphedema. Fibrosis and lymphangiogenesis are in opposite direction. While lymphangiogenesis is favorable for alleviating lymphedema, fibrosis is one of critical players in lymphedema. Both differ in terms of dependency on the VEGF-C/VEGFR-3 gradient. Fibrosis is a manifestation of excessive accumulation of extracellular matrix, mainly collagen, which subsequently harden the sites of wounds or injuries, inducing chronic inflammatory responses (Wynn, 2008). In contrast to lymphangiogenesis, that is, greatly suppressed in lymphedema and occurs due to the involvement of both VEGF-C and VEGFR-3, fibrosis was modulated in a VEGF-C-independent manner as demonstrated by Avraham et al. (2009) and earlier by Rutkowski et al. (2006), where only weak expression of VEGF-C was detected in the swollen tail of murine models (Avraham et al., 2009). Fibrosis is widely known as a key inhibitor of the tissue fluid accumulation which causes tissue expansion. This is a barrier to lymphangiogenesis (Avraham et al., 2009). It causes accumulation of collagen (Ghanta et al., 2015) and α-smooth muscle actin (α-SMA) around the podoplanin-expressing collecting lymphatic vessels (García Nores et al., 2018) takes a longer time than that of the interstitial fluid which results in a delayed lymphedema (Ghanta et al., 2015).

Fibrosis development is strongly associated with the M2-phenotype of macrophages (Ghanta et al., 2015) and Th2-skewed inflammatory response of CD4⁺ T cells (García Nores et al., 2018). M2-macrophages promote fibrosis by releasing transforming growth factor (TGF)-β1 as a profibrotic cytokine (Ghanta et al., 2015). In addition, they produce cytokines such as IL-13, IL-10, IL-4, tumor necrosis factor-α (TNF-α), and IL-1 (Van Linthout et al., 2014) which synergistically activate fibroblasts, recruit myofibroblasts, and exacerbate inflammatory cell infiltration to the injured site (Van Linthout et al., 2014; Ghanta et al., 2015). TNF-α, in particular, is shown to positively loop feedback the production of TGF-β1 via a kinase-specific pathway. However, the nuclear factor-κB (NF-κB) pathway is profoundly involved in the production of pro-inflammatory cytokines (Van Linthout et al., 2014). The significance of M2-macrophages in fibrosis is evident as their depletion ameliorates fibrosis. Notably, the depletion is selective and not general; during progressive injury, macrophage depletion leads to reduced fibrosis (Duffield et al., 2005), which is a favorable clinical feature in any of the proposed anti-lymphedema strategies. However, in the recovery state, this depletion is unable to overcome the recruitment of cellular and matrix components of the fibrotic response (Duffield et al., 2005), thus increasing fibrosis and accumulation of CD4⁺ T cells. In contrast, VEGF-C expression is weakened, suggesting macrophages as a source of VEGF-C along with LECs (Ghanta et al., 2015).

The CD4⁺ T cell subset plays a crucial role in fibrosis similar to that in M2-macrophages. This perspective was triggered following a critical finding by Nierdermeier et al. (2009) that fibrocyte differentiation was determined by the presence of CD4⁺ T cells. Fibrocytes are collagen-type I producing hematopoietic cells. Activation of the CD4⁺ T cell subset initiates the release of some soluble cytokines such as TNF, IL-4, IFN-γ, and IL-2, which prevent massive growth of fibrocytes. Interestingly, in the presence of calcineurin inhibitors and absence of mTOR inhibitors, when CD4⁺ T cells were in their activation state, marked collagen I deposition was detected, indicating the steady growth of fibrocytes. These results suggest that the activation of CD4⁺ T cells determines the fate of fibrocytes. This finding was supported by Tapmier et al. (2010), who demonstrated that depletion of CD4⁺ T cells using monoclonal antibodies was observed to significantly decrease the interstitial expansion and collagen deposition in a ureteric-obstructed RAG knock-out murine model setting. As such, CD4⁺ T cell blocking is considered a potential strategy for anti-fibrosis intervention. More recently, collagen III was also indicated to play a role in fibrotic skin of grade 3 lymphedema with the gene transcription level was detected much higher up to 39 fold than that seen for collagen I (Karayi et al., 2020). This bulks up potential targets of anti-lymphedema intervention although much more in-depth studies are necessitated to discern underlying mechanisms of both activation and regulation of collagen III from gene to protein levels and potential functional exchange between collagen I and collagen III.

In the global efforts to seek a cure for acquired lymphedema, the overall orchestration of fibrosis has recently received a lot of attention. Unlike targeting lymphangiogenesis, which not only allows re-building of the previously impaired lymphatic system but also re-promote cancer cell metastasis (Schlereth et al., 2014), targeting fibrosis is considered more appealing because it is an unfavorable prognostic factor for both cancer and lymphedema; hence, fibrosis suppression is expected to improve the outcomes for patients with cancer (Chandler et al., 2019) as well as for those suffering from lymphedema post cancer treatment (Gardenier et al., 2017; Mehrara et al., 2021).

One aspect that causes acquired lymphedema to be less likely, if not impossible, to cure is its progression cycle. The regions initially affected by lymphedema expand outwards to the skin through neighboring fatty tissues, resulting in concomitant changes in the lymphatic system and adipose tissue that must be focused to address lymphedema (Tashiro et al., 2017).

Adipose tissue is a lobulated septal network niched to diverse cell types, including adipocytes, fibroblasts, vascular endothelial cells (VECs), vascular smooth muscle cells, and immune cells mainly macrophages and lymphocytes (Tashiro et al., 2017). As represented by specialized organelles inside adipocytes, adipose tissue is responsible for the storage of triglycerides and cholesterol esters in the form of lipid droplets. Therefore, changes in the amount of lipids stored in adipocytes affect the fat cell size (DM et al., 2007). Adipocytes, the predominant cells residing in adipose tissue, are generated through a process termed adipogenesis, in which differentiation and maturation of adipocytes from mesenchymal stem cells (MSCs) are facilitated (Lane and Lennarz, 2013). This is a biphasic process and necessitates cell-cell communication via surface molecules, extracellular matrix, and direct contact. When preadipocytes completely develop into adipocytes, the cells use extracellular matrix to make envelops; thus, direct cell-cell contact is no longer possible (Schling and Löffler, 2002).

Transcriptional regulators modulate Adipogenesis. Zfp423 is a multi-zinc finger transcription factor that accounts for cell commitment to the adipogenic lineage (Gupta et al., 2012) and has exceptionally dense CpG sites in its promoter (Yang et al., 2013). This factor regulates peroxisome proliferator-activated receptor-γ (PPARγ), which is required for the formation of white and brown adipose tissue in the body, and CCAAT-enhancer-binding protein (C/EBPs) to facilitate differentiation of preadipocytes into adipocytes (Gupta et al., 2012). Zfp423 activation is facilitated via the bone morphogenetic protein (BMP)/Cae norhabditis elegans SMA and mothers against decapentaplegic (SMAD) signaling pathway (Bond et al., 2018) and the overall functions are mainly controlled by Wnt1-inducible signaling pathway protein 2 (WISP2), which forms a complex with the Zfp423 molecules in the cytoplasm. BMP/SMAD signaling enables dissociation of the WISP2-Zfp423 complex and Zfp423 enters the nucleus. Subsequently, transcriptional activation of some downstream genes is triggered (Yang et al., 2013; Bond et al., 2018; de sá et al., 2017).

In acquired lymphedema, adipogenesis is dramatically upregulated, causing the affected tissue to be suffused with adipocytes, further upregulating fat differentiation markers (Azhar et al., 2020) such as PPARγ, C/EBP-α/β, and FABP4 (Lowe et al., 2011; Ji et al., 2014; Roh et al., 2017a; Roh et al., 2020; Koc et al., 2021). Therefore, adipogenesis, along with other superficial clinical hallmarks, is a promising target for the treatment of lymphedema. These ideas have attracted increased attention after the achievement of successful treatment along with prolonged survival rate and improved prognosis by blocking or modifying adipogenesis activation-associated markers in some cases of cancer and obesity, particularly in women (Yang et al., 2013; Wang et al., 2017; Wu et al., 2019; Oshi et al., 2021).

Lymphatic filariasis is the most common pathophysiological condition caused by bacterial infection and characterized by impaired lymphatic transport affecting local immune response. Globally, more than 140 million people have been reported to suffer from lymphatic filariasis, mainly caused by Wuchereria bancrofti and less frequently by Brugia malayi (Kambhampati et al., 2016).

Interestingly, a recent case report by Hong et al. (2019) established a causative association between bacterial infection and lymphedema in the lower extremities of a patient with intestinal tuberculosis in Korea. Although an extremely rare case, it can broaden our horizon that lymphedema is not solely attributed to the adverse effects of anticancer regimens. Anti-tuberculosis drugs impaired lymphatic drainage, which resulted in ineffectiveness of the pertinent drug after the lymphatic system was damaged. These results were in line with those from previous studies by Hoda and Rab. (1974) in Korea. Both observed that although anti-tuberculosis drugs initially regressed lymph nodes and pulmonary lesions, lymphedema recurred after 7–9 months and persisted.

Obesity has been considered a strong risk factor for lymphedema (Savetsky et al., 2014; Nitti et al., 2016), causing a serious clinical issue (Shimizu et al., 2013). Obesity and lymphedema have been suggested to have a reciprocal interplay (Mehrara and Greene, 2014). This was clearly observed in adipose tissues isolated from lymphedema, which were identical to fat depots in obese patients; both results from the proliferation and hypertrophy of adipocytes, which subsequently leads to the infiltration of macrophages and lymphocytes, characteristic of chronic inflammation (Zampell et al., 2012; Mehrara and Greene, 2014). At the molecular level, C/EPB-α is an essential transcription factor for determining the fate of adipocyte lineage, and adiponectin, a peptide hormone that promotes differentiation of preadipocytes into adipocytes, is upregulated in the area proximal to the region of lymphatic obstruction. The upregulation of these two proteins decreases in the distal area, according to the gradient of lymphatic stasis (Aschen et al., 2012).

In the obese state, the plasma concentration of adiponectin is decreased. The functional role of adiponectin in the lymphatic system was well described in a mouse model with ablation of tail surface lymphatic network. In this model, adiponectin-KO mice showed a greater diameter of wounded tails than that of wild-type mice. Adiponectin deficiency was associated with a low number of LECs. This phenotype can be reversed by adiponectin administration, which restores the lymphatic vasculature. In vitro, adiponectin promotes the differentiation and viability of LECs through the AMP-activated protein kinase (AMPK)-Akt-endothelial nitric oxide synthase pathways (Shimizu et al., 2013).

In contrast to adiponectin, leptin has been shown to be upregulated in the obese state (Korda et al., 2008; Grossmann et al., 2010). It acts through the leptin receptor in the hypothalamus to suppress food intake, but obese patients are leptin-resistant (Grossmann et al., 2010). In addition to the brain, leptin exerts its biological actions on multiple peripheral tissues, including vascular and lymphatic endothelial cells (Grossmann et al., 2010).

In the context of angiogenesis, leptin mediates neovascularization by inducing the proliferation of vascular endothelial cells and remodeling the extracellular matrix through regulation of metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (Park et al., 2001).

A high concentration of leptin, as observed in obese patients, compromises lymphatic endothelial cell homeostasis, whereas a physiological concentration of leptin maintains cellular homeostasis. A high concentration of leptin induces the expression of the proinflammatory cytokine IL-6 in human dermal lymphatic endothelial cells. Furthermore, IL-6 rescues the compromised cell proliferation and tube formation caused by high-dose leptin treatment either in an autocrine or paracrine manner. This finding suggests that leptin and IL-6 are promising intervening agents to reduce the incidence of postoperative lymphedema (Sato et al., 2016).

Lipedema is linked to lymphedema, and both are often misdiagnosed (Peled and Kappos, 2016; Buso et al., 2019). This is a clinical manifestation of bilaterally symmetrical perturbed fat deposition in the lower or upper extremities with frequent ecchymosis, and can be triggered by a minor injury (Shavit et al., 2018); lipedema is a specific disease associated to fat distribution, a distinctive feature from lymphedema.

Lipedema affects approximately 18.8% of patients with enlargement of the lower limbs and is exclusively seen in women (Forner-Cordero et al., 2012). It has been reported that 6.5% of children diagnosed with lymphedema were actually suffered from lipedema (Szél et al., 2014). Moreover, approximately 60% of lipedema cases have been reported to be genetically acquired. Although lipedema-inducing genes are yet to be inherently studied (Grossmann et al., 2010), but studies by Makinen et al. and Harvey et al. have suggested the probable genes involved in it. Mäkinen et al. (2001) showed that VEGFR-3 missense mutation played a key role in the process of subcutaneous adipose tissue thickening, whereas. Harvey et al. (2005) demonstrated that the gene PROX1 was inactivated in an adult-onset obesity and lymphatic vascular disease mouse model. Notably, both genes have been studied for their relevance in lymphedema development, strongly suggesting molecular crosstalk between lymphedema and lipedema.

Lipedema has recently received increased attention, and efforts have been made to gain a comprehensive understanding of the pathological mechanism of the disease for the development of specific intervention strategies for lipedema distinguishable from those of lymphedema. We, hitherto, are left with a major query that whether lymphedema leads to lipedema or vice versa. Future studies should be directed to unveiling the molecular crosstalk between the two.