Molecules That Have Rarely Been Studied in Lymphatic Endothelial Cells

1. Introduction

The lymphatic vascular system was already known in ancient times, although its functions were misinterpreted. Venae albae or Ductus lactei were already known to Hippocrates of Kos (460-370 BC), Aristoteles (384 – 322 BC), and the doctors of the Alexandrian school (approx. 300 BC – 600 AC) (cited from [1]). And when Gaspare Aselli (re)discovered the lymphatics [2], he knew very well what he had to look for. He realized that the Venae albae were easy to find in the mesenteries e.g. of dogs, but he was probably the first to make a connection of these milky vessels with food intake. The anatomical representation of the lymphatic vascular system was especially advanced at Italian universities [3], although the function of the system was still very mysterious. One of the first to recognize the importance of directed fluid transport in the lymphatics was [4]. Contrary to the prevailing doctrine, which postulated a lymph flow into the liver, he described the connection to the central venous system. He also recognized a valve system in lymphatics and the coagulability of the lymph.

We should be very humble in evaluating ancient knowledge, because one thing is certain: we are still far from fully understanding the functions of the lymphatic vascular system, although more and more functions are being discussed [5,6,7,8]. The complexity is immediately apparent when we visualize the heterogeneity of lymphatic endothelial cells (LECs) [9], and the long lists of molecules expressed in LECs [10,11], lymph collectors [12] or lymph nodes [13,14]. Thereby, development and behavior of LECs is critically regulated by the transcription factor PROX1 [15,16,17]; and the histological characterization of human LECs is typically performed with antibodies against PROX1 and CD31 [18], the Vascular endothelial growth factor receptor-3 (VEGFR3, FLT4) [19], the Lymphatic vessel endothelial hyaluronic acid receptor 1 (LYVE1) [20], and the type-I integral membrane glycoprotein Podoplanin (PDPN) [21].

Congenital malformations of the blood vascular system are very common, those of the lymphatic system are much rarer, mostly located in the head-neck region and can be life threatening. Most of the lymphatic malformations are caused by somatic mutations in genes involved in the VEGFR3 signaling pathway [22,23,24,25]. However, most genetic causes of lymphatic malformations and primary lymphedema have not yet been discovered. This illustrates that there are still a very large number of molecules whose significance for the development and function of the lymphatic vessels is not yet known. We have previously used RNASeq to study human foreskin-derived LECs under normoxic and hypoxic conditions, and defined 162 genes that are significantly regulated by hypoxia [10], as well as highly expressed genes that influence the composition of the extracellular matrix and may be involved in lymphedema-induced fibrosis [26]. Here, we went through the RNASeq list and studied the large number of highly expressed LEC genes. We compared RNA expression with protein expression by systematically studying the Human Protein Atlas [27] (https://www.proteinatlas.org/). In this way, we sought to define additional molecules that can be used for deeper characterization of lymphatic vessels and to define further functions of LECs.

2. Results and Discussion

We recently performed expression analyses of three well characterized human foreskin-derived LEC lines under normoxic and hypoxic conditions [10,26], and received a list of approx. 16,000 LEC-expressed genes. Only a very small number of these molecules have been studied in LECs so far. We have tried to systematically match RNA expression with protein expression using the Human Protein Atlas [27]. We have concentrated on molecules whose function has not yet been investigated in LECs. However, the number of matches between RNA and protein expression was not very high, but there can be many technical reasons (fixation time of tissue, paraffine permeability of the antibodies, etc.) for this.

One of the main functions of the lymphatics is regulation of fluid homeostasis. The fluid is mainly absorbed via the flexible microvalves of the initial lymphatic vessels. However, part of the fluid uptake also occurs by means of active transcytosis, which has also been observed for the uptake of chylomicrons by lacteals [28,29,30]. Very recently the uptake of myofibroblast-derived microvesicles (MVs) was studied in human dermal BECs and LECs [31]. The authors describe that MVs cross an LEC layer but not a BEC layer in vitro. Internalization into BECs is mainly via the caveolin-dependent pathway, while LECs mainly use the macropinocytosis pathway. High expression of caveolin 1 (CAV1) and CAV2 (Table 1), for endo- and transcytosis, seems to be in line with the free passage of MVs through LECs; and antibodies against CAV1 and CAV2 clearly stain lymphatics, and selected blood vessels (Figure 1 and Figure 2).

Very active transcytosis by LECs is also reflected by the RNA expression the ferlin family members: myoferlin (MYOF; very high), dysferlin (DYSF; high), and otoferlin (OTOF; moderate) (Table 1). Ferlins regulate membrane fusion, and fusion of vesicles to cell membranes e.g. for exocytosis or membrane regeneration [32]. Of the three ferlins, we observed clear immunostaining against DYSF (dystrophy-associated fer-1-like protein) in various organs (Figure 3) and performed immunodouble staining with CD31 in human foreskin (Figure 4). We observed DYSF in dermal lymphatics. Blood vessels were mostly negative, except for the subepithelial capillary plexus. We tested various OTOF antibodies, but did not receive a positive result; and, in contrast to the high RNA expression, we found no immunopositivity for MYOF in the Human Protein Atlas. However, DYSF is clearly present in LECs, which is in line with high transcytotic activity.

Both the initial lymphatic vessels and the lymph collectors exhibit strong functional fluctuations in their diameter, and they have valves, whose elasticity is of great importance for reliable valve closure. Spectrins have originally been identified as the major elastic component of erythrocytes, linking the actin cytoskeleton to the cell membrane. However, nonerythroid spectrins have also been identified [33,34,35]. Spectrins form tetrameric proteins of alpha and beta subunits. SPTAN1 (spectrin, alpha, nonerythrocytic 1) and SPTBN1 (spectrin, beta, nonerythrocytic 1) are highly expressed in LECs (Table 1), and they are well detectable at protein level in lymphatics of various organs (Figure 5 and Figure 6). Neuropathies related to malfunction of the two molecules have been observed, however, lymphedema has not been described yet.

Among the genes most highly expressed in LECs is Multimerin 1 (MMRN1) (Table 1). Lymph contains fibrinogen and can coagulate [36], and we have previously pointed out that LECs are an important source for factor VIII (F8, antihemophilic globulin A) and its carrier protein von-Willebrand factor (VWF) [8]. MMRN1 is a specific coagulation factor V binding platelet protein with a role in hemostasis and coagulation, and accordingly it is highly expressed in megakaryocytes [37]. Besides that, it is found with extremely high specificity in endothelial cells, however, a clear distinction between BECs and LECs has not been made [38]. However, it appears that LECs are clearly stronger positive than BECs (https://www.proteinatlas.org/ENSG00000138722-MMRN1/single+cell+type). We found immunopositivity for MMRN1 in lymphatics of various organs (Figure 7). During lymphostasis, the accumulation of pro-coagulatory factors (F8, VWF, MMRN1) and the decrease in the anti-coagulatory and anti-inflammatory 5'-nucleotidase (CD73) of the LECs [39] can be important reasons for the increased tendency to thrombosis and inflammation.

The MARCKS-like protein-1 (MARCKSL1 = MLP) has great similarity with the myristoylated, alanine-rich protein MARCKS [40,41], a substrate for protein kinase C. MARCKSL1 has mainly been studied in neural development, and in cancer cell migration [42]. High MARCKSL1 expression has a strong prognostic value in lymph node-negative breast cancer patients [43]. Upon phosphorylation, MARCKSL1 induces actin bundling and inhibits cell migration [42]. We observed high RNA expression of MARCKSL1 in LECs (Table 1), and immune-positive lymphatics in various organs (Figure 8). The influence of MARCKSL1 on LEC stability and function has not been studied yet.

Another molecule associated with the actin cytoskeletal system is calponin 3 (CNN3). It consists of an acidic C terminus and a basic N terminus [44,45]. CNN3 regulates contractility of actomyosin-containing stress fibers of non-muscle cells [46]. Thereby, control of stress fiber contractility by CNN3 was found to be associated with mechanosensitive Yap/Taz (Yes-associated protein/transcriptional coactivator with PDZ binding motif) transcriptional activation [47]. We observed high RNA expression of CNN3 in LECs (Table 1) and immune-positive lymphatics in various organs (Figure 9), making LECs an attractive model for further studies on the function of CNN3. The importance of the Yap/Taz signaling in the Hippo pathway for lymphangiogenesis has been well recognized [48,49].

The cytoskeleton of LECs appears to be very stable, and accordingly, the invasiveness of LECs is usually extremely low; the only exception being the vanishing bone disease (Gorham-Stout disease, GHS) with approx. 350 cases being reported [50,51,52]. The ankyrin repeat domain protein 37 (ANKRD37) is not only associated with preeclampsia during pregnancy. Its knock-down enhances trophoblast invasiveness, migration, and regulation of key invasion proteins [53]. ANKRD37 is expressed at very low levels in LECs and significantly upregulated by hypoxia (Table 1) [26]. It is a hypoxia-inducible factor-1 (HIF1) target gene [54]. Its expression in GHS has, to the best of our knowledge, not been studied yet. We observed immune-positivity for ANKRD37 in LECs (Figure 10), however, due to the large number of ANKRD family members expressed in LECs (data not shown), specificity of antibody staining might be problematic.

Another molecule involved in actin stress fiber formation, and containing an ankyrin repeat domain, is KANK3 (KN motif- and ankyrin repeat domain-containing protein 3) [55]. It is highly expressed in LECs (Table 1), and immune-positivity is seen in lymphatics of various organs (Figure 11). The four members of the KANK family regulate integrin-mediated adhesion, actomyosin contractility, and link focal adhesions to the cortical microtubule stabilization complex [56]. Specific expression of KANK3 in endothelial cells has been noted [56], but functional studies are lacking.

Lymph contains high amounts of immunoglobulin G (IgG) heavy chain [36]. Antigen-IgG complexes represent a starting point for the classical complement system, which can stimulate phagocytes, inflammation and the cell-killing membrane attack complex (MAC) [57]. LECs express high amounts of CD59, aka protectin (Table 1), a glycoprotein functioning as a membrane-bound inhibitor of MAC [58]. Malfunctioning of CD59 causes hemolytic anemia with immune-mediated polyneuropathy [59]. The specific function in LECs has not been investigated yet. Immune-positivity is seen in lymphatics and blood vessels (Figure 12), suggesting an important immune-suppressive function in vessels. Strong expression of CD59 in colorectal cancer is associated with higher incidence of lymph node metastasis [55]. In ulcerative colitis, a chronic inflammatory disease of the colon, CD59 belongs to the complement components that have shown to safeguard the intestinal barrier and reduce intestinal inflammation [60].

It was previously shown that the non-canonical WNT (Wingless-type MMTV integration site) signaling pathway is of great importance for the elongation of lymphatics during embryonic lymphangiogenesis [61,62]. Nucleoredoxin (NXN) is a 48kDa protein and a redox-dependent negative regulator of the Wnt signaling pathway [63]. NXN mRNA is highly expressed in LECs (Table 1), and by immunohistology it can be detected in lymphatics of various organs (Figure 13). Similar to mutations in WNT5A [64] and other WNT signaling members, mutations in NXN cause the Robinow syndrome [65], which is mainly characterized by dysmorphic facial features and short-limbed dwarfism. To our knowledge, morphology and function of the lymphatics have not yet been investigated in Robinow patients.

3. Conclusion

The exome of lymphatic endothelial cells is very extensive, as in other cell types. The correspondence with the protein expression that we have studied in the Human Protein Atlas is comparatively low. However, this can probably be attributed to simple technical reasons when studying paraffin-embedded human tissues. We found good agreement for molecules associated with the high transcytosis activity of LECs (caveolin, dysferlin). There was very good agreement for molecules that have important functions in the structure and regulation of the cytoskeleton. This may be due to the fact that the LECs of the initial lymphatic vessels have a unique morphology and function. This is expressed in the oak leaf-like morphology of the cells with the formation of specialized button-like junctions and microvalves, which are of essential importance for the directed lymph flow [66,67,68]. Accordingly, we found expression of molecules that mediate cellular elasticity (spectrins) and interact with actin microfilaments (ANKRD37, CNN3, KANK3, MARCKSL1). In terms of immune regulation, lymphatic vessels are a double-edged sword [8]. They are the main route for the emigration of leukocytes towards the lymph nodes. However, they can also have an immunosuppressive effect, which is reflected in the expression of CD59. The influence of LECs on coagulation, which has already been described several times, was confirmed in our investigations (multimerin1). The importance of the WNT signaling pathway for development and function of lymphatics still needs to be investigated in greater detail (NXN). In sum, we hope our studies can stimulate further studies on the complex morphology and functions of lymphatics.

4. Materials and Methods

4.1. Cell Culture

We used three well characterized human dermal lymphatic endothelial cell lines (PromoCell, Heidelberg, Germany). Cells and culturing were described before [10,26].

4.2. RNA Sequencing

RNASeq of defined lymphatic endothelial cells was performed as described [10,26].

4.3. Immunofluorescence (IF)

IF was performed on human foreskin derived from operations performed at the University Medical Center Goettingen (UMG). Specimens were fixed in 4% paraformaldehyde for 1h, embedded in tissue freeze medium and sectioned at 12µm. Studies were performed with the informed consent of the patients or their parents, and were approved by the ethics committee of the UMG (application no. 18/1/18). Primary antibodies were: mouse-anti-human CD31(BD Pharmingen, dilution 1:50), rabbit-anti-human dysferlin (Sigma, dilution 1:200). Secondary antibodies were: Alexa 488-conjugated goat-anti-mouse IgG (H+L) (Invitrogen, dilution 1:200), and Alexa 594-conjugated goat-anti-rabbit IgG (H+L) (Invitrogen, dilution 1:200). Nuclei were counterstained with Dapi.

4.4. Immunohistochemistry

We compared our RNASeq expression data with protein expression by systematically studying the Human Protein Atlas [27] (https://www.proteinatlas.org/). All figures shown here can be found and further studied at variable magnification in this repository.