Abstract

Molecular pathways involved in lymphedema: Hydroxytyrosol as a candidate natural compound for treating the effects of lymph accumulation

Matteo Bertellia, Aysha Karim Kianib, Stefano Paolaccic,*, Elena Manarad, Astrit Dautaje, Tommaso Beccarif, Sandro Michelinig. Journal of Biotechnology 308 (2020) 82–86

Lymphedema is a chronic accumulation of interstitial fluid due to inefficient lymph drainage. Major causes of lymphedema are malformations of lymphatic vessels, trauma, toxic damage and surgery. The swelling typically affects the limbs. Lymphedema may be primary, caused by genetic mutations and relatively rare, or secondary (acquired), due to external causes such as infections or surgery. Fluid accumulation induces pathological changes: activation of the inflammatory cascade, immune cell infiltration, tissue fibrosis, adipose accumulation. We focused on the inflammatory phenotype mediated by leukotriene B4, a lipid mediator of the inflammatory pathway, and the potential therapeutic effect of hydroxytyrosol. We conducted an electronic search in PubMed using “lymphedema”, “lymphedema pathway”, “hydroxytyrosol” as keywords. We found that lymphedema deregulates at least six molecular pathways and that hydroxytyrosol, a compound with antioxidant activity, can improve endothelial dysfunction, hemostatic and lipid profiles, and decrease oxidative stress and inflammation through inhibition of leukotriene B4 activity. This review is the first to highlight the possibility of using hydroxytyrosol to treat the secondary effects of lymphedema, especially inflammation. The possible effects of hydroxytyrosol on lymphedema should be tested in vitro and in vivo to find the best way to treat patients with lymphedema inorder to improve their health status.

Main findings

This study identified six main pathways involved in the pathology of lymphedema.

1. Immunological pathways.

Lymphedema patients often show impaired immune function which predisposes them to infections. Chronic stagnation of lymph in lymphedematous tissue restricts the movement of immunocompetent cells and eventually damages local immune surveillance.

a. T helper 2 cells

T helper 2 (Th2) cells play a significant role in the development of lymphatic dysfunction and fibrosis.

b. Macrophages.

Several research groups have established that macrophages are recruited in lymphedematous tissue. The cytokines produced by lymphatic vessels eventually activate macrophages and produce inducible nitric oxide synthase, which in turn reduces lymphatic vessel contraction (Szuba et al., 2002). Reduced lymphatic contraction leads to fluid accumulation, impaired transport of antigens to regional lymph nodes, exacerbation of fibrosis and infiltration of CD4+ cells (Ghanta et al., 2015). Macrophages are recruited soon after lymphatic injury (Rutkowski et al., 2006), inducing dermal lymphangiogenesis through secretion of lymphangiogenic growth factors, such as VEGF-C.

2. Toll-like receptor pathway.

Toll-like receptors (TLRs) belong to a protein class that plays a pivotal role in the immune system. After lymphatic injury, reduced expression of TLRs aggravates lymphedema and inflammation (Zampell etal.,2012a),while lymphatic repair is reduced by expression of TLR-2, -4, and -9 (Zampell et al., 2012a). In patients with chronic lymphatic pathology, stimulation induced by three TLRs, TLR-2, -7, and -9, results in sharply increased production of pro-inflammatory cytokines (Babu et al., 2011).

3. Pro-inflammatory pathways

Inflammation is considered a major component in the pathophysiology of lymphedema (Ly et al., 2017). Several studies observed that most symptoms associated with lymphedema depend on expression of pro-inflammatory genes (Földi et al., 2000; Fu et al., 2016; Tabibiazar et al., 2006).

Inchroniclymphedema,almost70%ofinflammationiscausedbyT cells through a combined response of T-helper 1 (Th1) and Th2 cells.

4. Fibrosis

Accumulation of protein-rich lymph induces fibrosis in affected tissues. Fibrotic tissues further reduce lymph drainage (Markhus et al., 2013). Fibrosis is caused by infiltration of Th1 and Th2 cells in lymphedematous tissue (Ogata et al., 2016). Macrophages are also important in the regulation of fibrosis. Experimental studies have demonstrated that fibrosis occurs when accumulation of macrophages is inhibited (Ghanta et al., 2015).

5. Fat deposition

Deposition of adipose tissue is a prominent feature of lymphedema and may be triggered by minor dysfunction or injury of the lymphatic system (Cuzzone et al., 2014). Inflammation and fat deposition are closely linked in lymphedema. Enhanced IL-6 expression is inversely correlated withfat deposition: forexample,loss ofIL-6 linked to lymphatic injury leads to a sharp elevation in fat deposition (Cuzzone et al., 2014).

6. Oxidative stress pathway

Very few studies have investigated the role of oxidative stress pathways in lymphedema. A comparison of lymphedema patients and healthy controls revealed higherlevels ofreactive oxygen species (ROS) and enhanced lipid peroxidation in lymphedematous tissue.

Leukotriene B4 and its role in lymphedema

Leukotriene B4 (LTB4) is an important inflammatory lipid mediator in the pathogenesis of lymphedema (Nakamura et al., 2009). Leukotrienes are biologically active lipids, originally isolated from leukocytes. They are arachidonic acid metabolites produced by pro-inflammatory immune cells such as dendritic cells, macrophages, eosinophils, mast cells and neutrophils. These lipid mediators produce strong inflammatory responses by binding and activating their cognate G protein-coupled receptors (Nakamura et al., 2009). Several studies have established that LTB4 is implicated in the pathogenesis of lymphedema.

Hydroxytyrosol as a candidate natural compound for the treatment of the symptoms of lymphedema

Hydroxytyrosol (HT), a biophenol extracted from olives and also present in extra virgin olive oil, is a small molecule that quickly penetrates tissues and exists stably in free form or as a derivative of secoiridoid or acetate (Bonetti et al., 2016; Hu et al., 2014). A number of studies have established that HT and its derivatives have anti-inflammatory, antioxidant and antimicrobial activities that prevent oxidative stress (Burattini et al., 2013). Bioavailability studies showed that HT is absorbed in a dose-dependent manner in humans and animals after consumption of olive oil (Hu et al., 2014). Since its safety profile is excellent and it does not cause side-effects at any concentration.

Hydroxytyrosol as leukotriene B4 synthesis inhibitor

Inhumans,HTinhibits5-LOenzymeactivity inleukocytes,blocking leukotriene B4 generation, as demonstrated in several studies. The activity of HT in the inhibition of LTB4 synthesis is particularly important in lymphedema because LTB4 has the ability to diminish the function of the lymphatic endothelial cells, worsening the lymphatic vascular dysfunction, and favoring the disease progression (Jiang et al., 2018). The ability of HT to block 5-LO activity, thus suppressing generation of LTB4, provides a safe option for preventing and treating inflammation and for studying the therapeutic benefits in lymphedema patients (Tian et al., 2017).

Hydroxytyrosol and its role against fat deposition

HT also has anti-adipogenic effects. It induces apoptosis in preadipocytes, blocks visceral pre-adipocyte differentiation, and stimulates lipolysis in pre- and mature adipocytes (Stefanon et al., 2015).

Methods for the extraction of hydroxytyrosol

HT can be extracted from olive pomace using the ultrasound-assisted extraction, a fast, effective, environmentally friendly technique for extracting HT from olive pomace (Xie et al., 2019). Another possible method of extraction involves the infusion of chopped leaves in microfiltered water (Benincasa et al., 2019). HT can also be extracted from olives and/or from the solid residues of olives after the extraction of olive oil by acid hydrolysis and by purifying the obtained solution on a column containing acid activated anion exchange, and a column containing an adsorbent non-ionic resin; both columns being eluted with water to recover the HT (Lopez Mas et al., 2008).

A project for future research is to extract HT from natural sources like olives or olive leaves and use it pure, or in combination with other molecules with similar effects, for the treatment of symptoms caused by lymphedema.