Dietary long-chain fatty acids (FAs) and monoacylglycerols are absorbed on the apical membrane of enterocytes, repackaged into triglyceride (TG)-rich lipoproteins, or chylomicrons (CMs), and secreted into the intestinal lymphatic vessels, referred to as lacteals (Tso and Balint, 1986; Kohan et al., 2010; Mansbach and Siddiqi, 2010; Cifarelli and Abumrad, 2018). Recent reports have shown that chylomicron trafficking into lacteals is regulated by vascular endothelial growth factors (VEGFs), hormones, transcription factors and requires activation of specific signaling pathways (Van Dyck et al., 2007; Dixon, 2010; Choe et al., 2015; Bernier-Latmani and Petrova, 2017; Zhang et al., 2018) challenging the view of gut lymphatic endothelium being a passive barrier.

Defective growth of lymphatic vessels, or lymphangiogenesis, in the lacteals impairs lipid absorption and metabolism, as reported in several mice models of dysfunctional gut lymphatics due to genetic deletion of prospero homeobox gene Prox1 (Harvey et al., 2005), VEGF-C (Nurmi et al., 2015), Notch ligand delta-like ligand 4 (DLL4) (Bernier-Latmani et al., 2015), and adrenomedullin (Davis et al., 2017). VEGF-C, a major lymphangiogenic factor during embryonic development (Makinen et al., 2001; Karkkainen et al., 2004), regulates maintenance and function of intestinal lymphatics in adult mice (Nurmi et al., 2015) by binding its cognate tyrosine kinase VEGF receptor 3 (VEGFR-3), highly expressed in lymphatic endothelial cell (LECs). Upon binding with VEGF-C, VEGFR-3 forms homodimers (R3/R3) and undergoes intrinsic autophosphorylation on at least five cytoplasmic tyrosine residues (Dixelius et al., 2003). VEGFR-3 activation leads to protein kinase C-dependent activation of ERK1/2, implicated in cell proliferation (Pajusola et al., 1994; Fournier et al., 1995). VEGF-C can also bind to VEGFR-2, inducing formation of heterodimers between VEGFR-3 and VEGFR-2 (R3/R2) through a different autophosphorylation pattern (Dixelius et al., 2003; Nilsson et al., 2010). Postnatal deletion of Vegfc in mice leads to lacteal regression, defective lipid absorption and protects from diet-induced obesity and insulin resistance (Nurmi et al., 2015), without affecting any other lymphatic beds. Similar to Vegfc deletion, genetic ablation of Vegfr3 in adult mice affects lacteal maintenance and function (Nurmi et al., 2015) although impact in lipid absorption was not investigated. Whether VEGFR-3 signaling affects TG absorption and distribution remains unclear. Here we investigate the role of VEGFR-3 signaling in TG trafficking from enterocyte into the circulation. We used the Chy mutant mouse, which carries an inactivating mutation in VEGFR-3 tyrosine kinase domain (heterozygous A3157T mutation resulting in I1053F substitution). Chy mutant mice present defective lymphatic vessels and develop lipid-rich chylous ascites at birth that resolves upon weaning (Karkkainen et al., 2001). To our knowledge, the mechanisms through which inactivation of VEGFR-3 signaling pathways affects lipid absorption and trafficking to blood circulation remains unknown.

Our data show the novel finding that VEGFR-3 signaling regulates absorption and trafficking of dietary lipids from the enterocytes to the circulation. VEGFR-3 is initially expressed in all embryonic endothelia, but becomes highly restricted to the lymphatic endothelium in adult tissues (Kaipainen et al., 1995). However, VEGFR-3 can be found at sites of inflammation and tumor in non-endothelial bone marrow-derived cells (Skobe et al., 2001), stromal dendritic cells (Hamrah et al., 2004) and some VEGFR-3 expression has been described in angiogenic blood endothelial cells (Valtola et al., 1999). The Chy mutant mouse model carries an inactivating mutation in the tyrosine catalytic domain of VEGFR-3. We show that Chy mice have markedly decreased postprandial levels of TGs in the circulation, as compared with controls. Since lipoprotein lipase (LPL) activity was inhibited as previously reported (Otway and Robinson, 1967), the decrease in postprandial TG levels mainly reflects input from the small intestine. Surprisingly, cholesterol levels were also significantly decreased in plasma of Chy mice compared with littermate controls (Figure 1). The significance of this change is unclear and might reflect abnormality of systemic cholesterol metabolism. The lymphatic vasculature regulates removal of cholesterol from peripheral tissues (Lim et al., 2013; Randolph and Miller, 2014) and defective lymphatics impair reverse cholesterol transport (Martel et al., 2013).

Distribution of TGs in different sections of the intestine was strikingly altered in the Chy mouse. Under unchallenged 12-h fasting conditions, there was significant accumulation of TGs in the middle and distal sections of the Chy mouse as compared with control group (Figure 2). Following intragastric administration of triolein, intestines of control mice showed TGs accumulation in the proximal small intestine at 120 min post triolein gavage, about doubled those in fasting. However, in the Chy mouse, TG accumulation (∼threefold compared with fasting levels) was observed throughout the length of the small intestine (Figure 2). This was consistent with impaired proximal TG absorption resulting in more TGs reaching the middle and distal sections. In line with this, enterocytes contained larger cytoplasmic lipid droplets, visualized by Perilipin 3 staining supporting a defect in transfer of the TGs from the enterocyte to the lymphatic system and resulting in increased lipid (TGs and FFAs) excretion in the feces of Chy mice as compared with controls (Figure 2C). VEGF-C in the gut is manly produced by smooth muscle cell and muscle fibers surrounding the lacteals (Nurmi et al., 2015). Postnatal deletion of Vegfc causes atrophy of the lacteal, impairs lipid absorption and associates with increased lipid excretion in the feces (Nurmi et al., 2015). In the case of the Chy mouse, the intestinal lymphatics are enlarged (Karkkainen et al., 2004) and our data suggest that they are not functioning in TG transport. Thus, VEGFR-3 signaling in the small intestine appears important during lipid absorption. Individuals with hereditary or primary lymphedema (Milroy’s disease) due to an inactivating mutation in VEGFR-3 kinase domain (Irrthum et al., 2000) often present steatorrhea, which reflects the failure of intestinal lymphatics to adequately transport chylomicrons, with chylous ascites and lymphopenia due to loss of lymph retention (Lee and Young, 1953; Gerstein et al., 1957; Alexander et al., 2010). Retention of chylomicrons in the enterocytes is reported in individuals carrying mutations in the SAR1B gene that encodes for sar1b, a GTP-binding protein important for chylomicron trafficking in the enterocytes (Cifarelli and Abumrad, 2018); however, the lymphatic function in these individuals is not affected. VEGFR-3 is highly expressed in LECs and upon binding VEGF-C it dimerizes and undergoes autophosphorylation leading to activation of ERK1/2. It also heterodimerizes with VEGFR-2 leading to activation of AKT (Pajusola et al., 1994; Dixelius et al., 2003; Nilsson et al., 2010). Although VEGF-C can still signal via VEGFR-2 it is possible that the magnitude of VEGF-C/VEGFR-2 signaling might be altered by absence of a functional VEGFR-3. Zhang et al. (2018) have elegantly shown that chylomicron uptake by the lacteals is regulated by dynamic changes of cell-cell junctions via VEGF-A signaling through VEGFR-2. Zippering of lacteal junctions prevents chylomicron uptake and protects mice from diet-induced obesity (Zhang et al., 2018). It is possible that inhibition of VEGFR-3 signaling in the Chy mouse increases VEGFR-2 signaling leading to inhibition of TG uptake by lacteals.

The retention of TGs in enterocytes of the Chy mouse suggests that VEGFR-3 signaling might be involved in promoting efficient TG transfer from enterocytes to lacteals. This is further suggested by our data showing reduced nitric oxide generation in the Chy mouse (Figure 2D). Nitric oxide regulates mobilization of postprandial chylomicrons from the enterocytes into the bloodstream (Hsieh et al., 2015). Disruption of this pathway could be responsible for the accumulation of TGs observed in the enterocytes and mucosa and the subsequent lipid excretion into the feces. Furthermore, VEGF-C binding to VEGFR-3 selectively phosphorylates endothelial nitric oxide synthase (eNOS) at Ser 1177 via the PI3K/Akt pathway in LECs and this effect is not mediated via VEGFR-1 and/or VEGFR-2 (Coso et al., 2012). We hypothesized that generation of nitric oxide followed by VEGF-C binding to VEGFR-3 might affect lacteal permeability, favoring mobilization of TGs from the enterocytes into the lacteals, as it has been proposed (Hsieh et al., 2015). In summary, our study show the novel finding that VEGFR-3 signaling is an important regulator of dietary TG absorption and retention in the enterocytes possibly via generation of NO.

TS, NW, and VC ran the experiments and collected and analyzed the data. VC designed the work and wrote the manuscript. All authors critically revised and approved the manuscript.

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.