Cooperation of Complement MASP-1 with Other Proinflammatory Factors to Enhance the Activation of Endothelial Cells

Endothelial cells play an important role in sensing danger signals and regulating inflammation. Several factors are capable of inducing a proinflammatory response (e.g., LPS, histamine, IFNγ, and bradykinin), and these factors act simultaneously during the natural course of the inflammatory reaction. We have previously shown that the complement protein mannan-binding lectin-associated serine protease-1 (MASP-1) also induces a proinflammatory activation of the endothelial cells. Our aim was to investigate the possible cooperation between MASP-1 and other proinflammatory mediators when they are present in low doses. We used HUVECs and measured Ca2+ mobilization, IL-8, E-selectin, VCAM-1 expression, endothelial permeability, and mRNA levels of specific receptors. LPS pretreatment increased the expression of PAR2, a MASP-1 receptor, and furthermore, MASP-1 and LPS enhanced each other’s effects in regulating IL-8, E-selectin, Ca2+ mobilization, and changes in permeability in a variety of ways. Cotreatment of MASP-1 and IFNγ increased the IL-8 expression of HUVECs. MASP-1 induced bradykinin and histamine receptor expression, and consequently, increased Ca2+ mobilization was found. Pretreatment with IFNγ enhanced MASP-1-induced Ca2+ mobilization. Our findings highlight that well-known proinflammatory mediators and MASP-1, even at low effective doses, can strongly synergize to enhance the inflammatory response of endothelial cells.


Introduction
The endothelial lining of our circulatory system plays a role in several important physiological processes by regulating the blood pressure, hemostasis, and leukocyte homing and controlling vascular permeability. Endothelial cells (ECs) act as signal integrators; in other words, they have numerous cell surface receptors, adhesion molecules, and a welldeveloped cytoskeleton system to respond adequately to various molecules present in the blood [1]. As they are in direct contact with blood, the activation of plasma cascade systems, cytokines, chemokines, proteases, and other molecules produced by immune and non-immune cells in the blood directly and simultaneously affect their behavior. These factors belong to different parts of the immune system, and their combined presence and/or activation are characteristic of different diseases and pathological conditions.
Histamine is the major mediator of acute allergic reactions, released from mast cells and basophil granulocytes [14]. Histamine acts on four receptors (H1R-H4R) with different tissue distributions and effects, but most notably, in the vascular system, it rapidly increases endothelial permeability through H1R [15,16].
IFNγ is a cytokine produced predominantly by T H 1, T C 1, and NK cells during viral infections. IFNγ has two receptors (IFNGR1 and 2), and it is an important activator of macrophages and an inducer of Class II major histocompatibility complex (MHC) molecule expression. It activates the inducible nitric oxide synthase (iNOS), promotes adhesion and leukocyte migration, and increases endothelial permeability [21].
In most in vitro models, the response elicited by a single selected molecule is usually studied. However, the effects of this molecule can be substantially modified if we apply it with a combination of several other relevant substances, and the resulting cellular response can be significant even in low individual concentrations [27]. To model the cooperation of MASP-1 with molecules that have fundamental roles in microbial-, allergic-, and other complement systems involving diseases, we chose four important endothelial cell activators (LPS, IFNγ, bradykinin, and histamine) with different effects. ECs express the receptors for the above-mentioned activators, creating the possibility that we can assess the effects of the potential interaction between these molecules and MASP-1. The cooperation can manifest in several ways, such as the altered expression of cytokines and adhesion molecules, increased or decreased receptor expression, and distinct intracellular signaling or altered permeability.
In a major injury, the activation of the complement pathway and the entry of bacteria or viruses occur almost simultaneously (cotreatment). In a milder infection, when bacteria break through a tissue layer, the activation of the complement pathway in the blood can be a secondary reaction (pretreatment with a coactivator). However, in chronic or prolonged inflammation, the lectin pathway may already be activated, and a secondary infection may result in newly synthesized or incoming activators (pretreatment with MASP-1). The investigation of this cooperation may contribute to a better understanding of the behavior of endothelial cells during inflammatory conditions.
In this study, we present that MASP-1 interacts synergistically with other EC activator molecules to induce an increased proinflammatory response in endothelial cells.

Experimental Setup
Different proinflammatory activators acting on endothelial cells (among others) can be present simultaneously or sequentially in the circulation during a bacterial (LPS and MASP-1) or viral (IFNγ and MASP-1) infection, in certain diseases such as HAE (bradykinin and MASP-1), or during an allergic reaction accompanied by a bacterial or fungal infection (histamine and MASP-1). Three types of possible interactions were investigated to explore possible cooperation with the complement lectin pathway and other coactivators. Cells were either cotreated with recombinant MASP-1 (rMASP-1) and one of the coactivators, pretreated with rMASP-1 and then treated with one of the coactivators, or treated with rMASP-1 after pretreatment with one of the coactivators. These experimental setups modeled different pathophysiological conditions. The pretreatments always lasted for 24 h, while treatment time varied depending on the parameter measured ( Figure 1). Pre-determined [7] suboptimal doses of treatments were used to ensure the possibility of an accurate measurement of the cellular response, even when combined treatments were applied. In this study, we present that MASP-1 interacts synergistically with other EC activator molecules to induce an increased proinflammatory response in endothelial cells.

Experimental Setup
Different proinflammatory activators acting on endothelial cells (among others) can be present simultaneously or sequentially in the circulation during a bacterial (LPS and MASP-1) or viral (IFNγ and MASP-1) infection, in certain diseases such as HAE (bradykinin and MASP-1), or during an allergic reaction accompanied by a bacterial or fungal infection (histamine and MASP-1). Three types of possible interactions were investigated to explore possible cooperation with the complement lectin pathway and other coactivators. Cells were either cotreated with recombinant MASP-1 (rMASP-1) and one of the coactivators, pretreated with rMASP-1 and then treated with one of the coactivators, or treated with rMASP-1 after pretreatment with one of the coactivators. These experimental setups modeled different pathophysiological conditions. The pretreatments always lasted for 24 h, while treatment time varied depending on the parameter measured ( Figure 1). Pre-determined [7] suboptimal doses of treatments were used to ensure the possibility of an accurate measurement of the cellular response, even when combined treatments were applied. We measured cellular responses that were previously shown to be affected by MASP-1, either at the mRNA or protein level in endothelial cells. Intracellular Ca 2+ mobilization led to general signaling from various activated receptors, including G-protein-coupled protease-activated receptors (PARs), released IL-8 acted as a neutrophil chemoattractant, and adhesion molecules, such as E-selectin and VCAM-1, promoted the rolling and We measured cellular responses that were previously shown to be affected by MASP-1, either at the mRNA or protein level in endothelial cells. Intracellular Ca 2+ mobilization led to general signaling from various activated receptors, including G-protein-coupled protease-activated receptors (PARs), released IL-8 acted as a neutrophil chemoattractant, and adhesion molecules, such as E-selectin and VCAM-1, promoted the rolling and transmigration of leukocytes on endothelial cells, whereas increased vascular permeability ensured an effective extravasation of soluble immunological and acute phase mediators.
Due to the large amount of data generated, in the following section, only those results are presented in detail, where significant cooperation could be found between MASP-1 and the various treatments. Nevertheless, we summarized the data obtained from every experimental setup in Table 1. Table 1. Cooperation between rMASP-1 and other coactivators. Various combinations of pretreatments and cotreatments were applied on the HUVECs, and several indicators of the endothelial cell activation were measured. An interaction was considered significant if the measured outcome of the combined treatment was statistically greater or smaller than the effect of each activator alone in the same experimental setup. All significant interactions (p < 0.05) that we found were positive (indicated by green coloring), and their directions are indicated by arrows. Ca 2+ mobilization is an important step in the signal transduction of several receptors, such as GPCR-coupled histamine, bradykinin receptors, and PARs. Although LPS or IFNγ themselves did not trigger Ca 2+ mobilization in HUVECs, pretreatment with either of these factors significantly increased the Ca 2+ mobilization response to rMASP-1 treatment ( Figure 2A). Interestingly, cotreatment with MASP-1 with either BK or HA did not result in an increased Ca 2+ mobilization however, both BK and HA elicited greater Ca 2+ mobilization in the HUVECs after rMASP-1 pretreatment ( Figure 2B). an increased Ca 2+ mobilization however, both BK and HA elicited greater Ca 2+ mobilization in the HUVECs after rMASP-1 pretreatment ( Figure 2B).

rMASP-1 Cooperates with LPS in the Induction of E-selectin Expression
We measured the expression of two important adhesion molecules, E-selectin and VCAM-1, indicating proinflammatory changes in the adhesion capacity of endothelial cells to leukocytes. LPS and rMASP-1 interacted in the induction of E-selectin in two different ways. Both the cotreatment and the LPS pretreatment were followed by rMASP-1 treatment and significantly increased the E-selectin levels in HUVECs ( Figure 3A,B). We could not find any significant cooperation in the induction of VCAM-1 between rMASP-1 and the other activators.  In the case of cotreatment, LPS (10 ng/mL) and rMASP-1 (0.6 µM) were added together to the cells for 6 h. Then, the cells were fixed, and the expression level of E-selectin was determined by cell-based ELISA. Compared to the control: control vs. rMASP-1: ns., control vs. LPS-pretreated control: ns., control vs. rMASP-1 treatment after LPS pretreatment: ****, control vs. LPS: ****, control vs. LPS + rMASP-1: ****; +: indicates that the pretreatment or treatment was added; -: indicates that pretreatment or treatment was not added; ns: non-significant; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

rMASP-1 Cooperates with LPS, IFNγ, and Histamine in the Induction of IL-8 Production
IL-8 is a chemokine secreted by endothelial cells with a low constitutive rate and can be induced by various stimuli. Treatment with LPS [28], histamine, or rMASP-1 [7] alone increased the expression of IL-8, whereas IFNγ had no such effect on HUVECs (Figure 4). Cotreating the cells with rMASP1 and LPS or rMASP-1 and histamine significantly increased the yield of IL-8 secretion, compared to the corresponding individual treatments. Interestingly, cotreatment with rMASP-1 and IFNγ also significantly increased the IL-8 production of HUVECs ( Figure 4). IL-8 is a chemokine secreted by endothelial cells with a low constitutive rate and can be induced by various stimuli. Treatment with LPS [28], histamine, or rMASP-1 [7] alone increased the expression of IL-8, whereas IFNγ had no such effect on HUVECs (Figure 4). Cotreating the cells with rMASP1 and LPS or rMASP-1 and histamine significantly increased the yield of IL-8 secretion, compared to the corresponding individual treatments. Interestingly, cotreatment with rMASP-1 and IFNγ also significantly increased the IL-8 production of HUVECs ( Figure 4).  . rMASP-1 interacted with IFNγ, histamine, and LPS in the induction of the IL-8 production of HUVECs. Confluent layers of HUVECs were cultured in 96-well plates, and the cells were cotreated with rMASP-1 and LPS, histamine, or IFNγ for 24 h. The supernatants were collected and diluted in 1:20, and then the IL-8 concentration was measured by sandwich ELISA. The results of the ELISAs were calculated from the standard curve and plotted as mean concentration values of three independent experiments. Compared to the control: control vs. rMASP-1: ****, control vs. IFNγ: ns., control vs. histamine: ****, control vs. LPS: ****; +: indicates that the pretreatment or treatment was added; -: indicates that pretreatment or treatment was not added; ns: non-significant; *** p < 0.001; **** p < 0.0001.

rMASP-1 and LPS Cooperates in Endothelial Permeability Induction
To measure the intensity of paracellular transport through the endothelial layer, we used a modified version of the XperT technique [9,29]. Both 24 h rMASP-1 and LPS treatments themselves increased permeability, but the combination of the two treatments further opened the endothelial layer ( Figure 5). IFNγ: ns., control vs. histamine: ****, control vs. LPS: ****; +: indicates that the pretreatment or treatment was added; -: indicates that pretreatment or treatment was not added; ns: non-significant; *** p < 0.001; **** p < 0.0001.

rMASP-1 and LPS Cooperates in Endothelial Permeability Induction
To measure the intensity of paracellular transport through the endothelial layer, we used a modified version of the XperT technique [9,29]. Both 24 h rMASP-1 and LPS treatments themselves increased permeability, but the combination of the two treatments further opened the endothelial layer ( Figure 5).

mRNA Measurements for Receptor Expression
One of the most straightforward modes of cooperation is that pretreatment increases the expression of the receptor for the factor used subsequently. To assess this effect, we measured the expressions of histamine (HRH1), bradykinin (BDKRB1,2), and MASP-1 (PARs 1, 2, and 4) receptors, as the responses given to these were significantly increased by certain pretreatments. As we demonstrated, rMASP-1 pretreatment potentiated the bradykinin-induced Ca 2+ mobilization in HUVECs ( Figure 2B). Along with this, rMASP-1 significantly increased the mRNA levels of both B1 and B2 bradykinin receptors (BDKRB1 and 2) ( Figure 7A). rMASP-1 also increased the sensitivity of endothelial cells to histamine ( Figure 2B), and we found a tendency for elevated mRNA levels in the H1 histamine receptor after a 2 h long rMASP-1 treatment, although it was not statistically significant ( Figure 7B).
LPS pretreatment potentiated several rMASP-1-induced cellular responses (Ca 2+ mobilization, E-selectin expression, and permeability); therefore, we measured the mRNA Figure 6. LPS pretreatment followed by rMASP-1 treatment increased endothelial permeability. Confluent layers of HUVECs were cultured in 96-well plates precoated with biotinylated gelatin. The cells were pretreated with LPS (10 ng/mL) or with medium alone (control) for 24 h, and then rMASP-1 treatment (0.6 µM) was added for 20 min. Thrombin (300 nM, 20 min) was used as a positive control. The cell-free areas of the biotinylated gelatin surface were stained with Streptavidin-Alexa488, and after fixation, fluorescence was detected with a fluorescence plate reader; representative images were taken using an Olympus IX-81 fluorescence microscope. (A) Representative images of three independent experiments. The scale bar applies to all photos. (B) Mean values of three independent experiments normalized to the controls. Compared to the control: control vs. LPS-pretreated control: ****, control vs. rMASP-1: ***, control vs. LPS-pretreated rMASP-1: ****, control vs. thrombin: ****; +: indicates that the pretreatment or treatment was added; -: indicates that pretreatment or treatment was not added; *** p < 0.001; **** p < 0.0001.

mRNA Measurements for Receptor Expression
One of the most straightforward modes of cooperation is that pretreatment increases the expression of the receptor for the factor used subsequently. To assess this effect, we measured the expressions of histamine (HRH1), bradykinin (BDKRB1,2), and MASP-1 (PARs 1, 2, and 4) receptors, as the responses given to these were significantly increased by certain pretreatments. As we demonstrated, rMASP-1 pretreatment potentiated the bradykinin-induced Ca 2+ mobilization in HUVECs ( Figure 2B). Along with this, rMASP-1 significantly increased the mRNA levels of both B1 and B2 bradykinin receptors (BDKRB1 and 2) ( Figure 7A). expression of the three protease-activated receptors that could be activated by MASP-1. We found a strong increase in the PAR2 mRNA levels after a 24 h LPS treatment (FC: 6.46), whereas the levels of the other two receptors remained unchanged ( Figure 7C). Besides LPS, IFNγ also increased rMASP-1-induced Ca 2+ mobilization. We found interesting changes in the pattern of PAR receptor expression. The expression of PAR2 was downregulated (2 h FC: 0.77, p < 0.01; 24 h FC: 0.49, p < 0.001), while the expression of PAR4 slightly decreased after 2 h (FC: 0.59, p < 0.05), then slightly increased after 24 h (FC: 1.58; although it did not reach statistical significance, the tendency was clear) ( Figure 7D).

Discussion
Here, we report for the first time that the complement serine protease MASP-1 was able to cooperate with other activators to induce various pro-inflammatory responses in endothelial cells. We showed that the intracellular Ca 2+ mobilization, the expression of receptors, adhesion molecules and cytokines, and the increased permeability were significantly modified by diverse experimental setups (i.e., different timings and combinations of the activators).
On its own, MASP-1 induced intracellular Ca 2+ mobilization, the expression of E-selectin and IL-8, and increased the permeability of endothelial cells. One sample t-test was carried out to see if fold changes significantly differed from 1. Data from three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001; ns: not significant. rMASP-1 also increased the sensitivity of endothelial cells to histamine ( Figure 2B), and we found a tendency for elevated mRNA levels in the H1 histamine receptor after a 2 h long rMASP-1 treatment, although it was not statistically significant ( Figure 7B).
LPS pretreatment potentiated several rMASP-1-induced cellular responses (Ca 2+ mobilization, E-selectin expression, and permeability); therefore, we measured the mRNA expression of the three protease-activated receptors that could be activated by MASP-1. We found a strong increase in the PAR2 mRNA levels after a 24 h LPS treatment (FC: 6.46), whereas the levels of the other two receptors remained unchanged ( Figure 7C). Besides LPS, IFNγ also increased rMASP-1-induced Ca 2+ mobilization. We found interesting changes in the pattern of PAR receptor expression. The expression of PAR2 was downregulated (2 h FC: 0.77, p < 0.01; 24 h FC: 0.49, p < 0.001), while the expression of PAR4 slightly decreased after 2 h (FC: 0.59, p < 0.05), then slightly increased after 24 h (FC: 1.58; although it did not reach statistical significance, the tendency was clear) ( Figure 7D).

Discussion
Here, we report for the first time that the complement serine protease MASP-1 was able to cooperate with other activators to induce various pro-inflammatory responses in endothelial cells. We showed that the intracellular Ca 2+ mobilization, the expression of receptors, adhesion molecules and cytokines, and the increased permeability were significantly modified by diverse experimental setups (i.e., different timings and combinations of the activators).
On its own, MASP-1 induced intracellular Ca 2+ mobilization, the expression of Eselectin and IL-8, and increased the permeability of endothelial cells.
As we are the first to examine the cooperation between MASP-1 and other activators, there is no direct literature data available on this topic. Although information on the use of specific PAR agonists or thrombin is available, it is important to keep in mind that there are differences between MASP-1 and thrombin receptor usage. While thrombin is able to cleave PARs 1, 4, and possibly PAR3 but not PAR2 [30], MASP-1 cleaves PARs 1, 2, and 4 [5].
Cotreating HUVECs with MASP-1 and other activators showed that MASP-1 cooperated with LPS in the induction of IL-8 and E-selectin expression. In line with our findings, Ostrowska and Reiser showed, in airway epithelial cells, that the simultaneous LPS and PAR1 agonist, PAR2 agonist, or thrombin treatment increased the expression of IL-8 more strongly than separate treatments did [31]. This phenomenon may be due to the use of common signaling pathways. In our previous article, we showed that MASP-1-triggered IL-8 production was regulated predominantly by the p38-MAPK pathway [7]. We also showed that LPS induced the p38-MAPK pathway in HUVECs [28]. Besides the activation of p38-MAPK, Rallabandhi et al. showed that both the PAR2 agonist and LPS caused phosphorylation of ERK 1/2 and increased the IL-8 and tissue factor (TF) expression at the mRNA level in SW620 colonic epithelial cells [32]. Pan et al. found that LPS increased Il-8 production via the NFAT pathway in epithelial cells [33], and according to Jia et al., PAR2 receptor activation also promoted the nuclear translocation of NFAT [34].
Pretreatment with MASP-1 followed by adding LPS did not result in any significant changes in the measured responses. However, when we pretreated HUVECs with LPS and then added MASP-1, we observed significant cooperation in Ca 2+ mobilization, E-selectin expression, and the induction of permeability. Our receptor expression measurements at the mRNA level showed that LPS directly increased PAR2 receptor expression, possibly causing the stronger reactions observed. In line with our findings, Chao et al. found that LPS increased PAR2 expression, as well as Ca 2+ mobilization; MCP-1 expression and p38 phosphorylation were augmented in cells pretreated with LPS subsequently treated with trypsin [35]. As mentioned above, MASP-1 also activated the p38-MAPK pathway [7], creating the possibility for an additional cooperation at the level of the signaling pathways.
MASP-1 increased IL-8 production, whereas IFNγ treatment alone did not induce the expression of IL-8 in HUVECs. Beck et al. also found that IL-8 production was not affected by IFNγ treatment in HUVECs [36]. Interestingly, the combination of the two treatments significantly increased the production of IL-8. Similar to our results, Suk et al. found that IFNγ enhanced thrombin-induced IL-8 production in the human monocytic cell line U937 [37]. As there are no currently available data on the direct cooperation of the IFN and PAR receptors, we hypothesize that the cooperation occurs at the level of the signaling pathways and/or the transcription factor usage of the IL-8 gene promoter region. The promoter of IL-8 has binding sites for IFNγ-activated (e.g., IRFs, STAT1) and PAR receptor-activated transcription factors (e.g., NFAT, NFκB) [38,39].
Both MASP-1 and histamine alone increased the IL-8 production of the HUVECs. We found that MASP-1 and histamine cotreatment further induced this. Zhou et al. found that histamine induced IL-8 expression via the NFAT pathway in HUVEC [40]. As mentioned above, the activation of PAR2 also promoted the nuclear translocation of NFAT [34], so we hypothesize that the stronger induction of the NFAT pathway can be behind the observed, increased IL-8 production.
MASP-1 also affected the expression of BK receptors. We found an increase in the mRNA levels of both BK receptors (BDKRB1 and 2) after MASP-1 treatment and, in line with this, enhanced Ca 2+ -mobilization to BK.
We did not find a significant increase in the histamine receptor (HRH1) expression at the mRNA level after a 2 h long MASP-1 treatment, but there was a tendency towards it; in line with this, increased Ca 2+ -mobilization to histamine was observed after MASP-1 pretreatment. Since MASP-1 activates the NFκB pathway, and there are several NFκB binding sites in the promoter region of HRH1, it is possible that we can find higher mRNA levels at later time points.
IFNγ pretreatment increased the Ca 2+ mobilization in response to MASP-1 treatment. In concert with this, we found that IFNγ pretreatment significantly changed the expression pattern of PAR2 and PAR4 at the mRNA level. In line with our findings but using thrombin as a secondary stimulator, Watanabe et al. found that IFNγ pretreatment enhanced the Ca 2+ mobilization (and prostacyclin production) in HUVECs [41].
It is important to note that our study had limitations. Due to the large number of cooperations studied, we did not have the possibility to thoroughly investigate the possible mechanisms standing behind the observed interaction. We only relied on mRNA measurements and literature data when we tried to find an explanation for them. Our main goal was to identify the diverse types of cooperations and highlight the importance of the combined effect of various activators on the endothelial cells (Figure 8). A yellow background indicates pretreatment with a coactivator followed by rMASP-1 treatment, a pink background indicates rMASP-1 pretreatment followed by treatment with a coactivator, and a blue background indicates cotreatment. Inside the circle, we indicated possible ways of cooperation. The red text indicates results described in this article; black text indicates data from literature [7,28,34,35,38,39,40]. Blue oval: bradykinin receptors 1 and 2; red oval: PARs 1, 2, and 4; green oval: interferon-γ receptors 1 and 2; golden oval: histamine receptor 1; rose-colored oval: toll-like receptor 4.
In conclusion, here, we present the very first study investigating the cooperation of complement MASP-1 and other inflammatory factors in the induction of proinflammatory changes in endothelial cells. As endothelial cells play a substantial role in the regulation Figure 8. Complement MASP-1 and other proinflammatory factors cooperated in the induction of proinflammatory changes in endothelial cells. A yellow background indicates pretreatment with a coactivator followed by rMASP-1 treatment, a pink background indicates rMASP-1 pretreatment followed by treatment with a coactivator, and a blue background indicates cotreatment. Inside the circle, we indicated possible ways of cooperation. The red text indicates results described in this article; black text indicates data from literature [7,28,34,35,[38][39][40]. Blue oval: bradykinin receptors 1 and 2; red oval: PARs 1, 2, and 4; green oval: interferon-γ receptors 1 and 2; golden oval: histamine receptor 1; rose-colored oval: toll-like receptor 4.
In conclusion, here, we present the very first study investigating the cooperation of complement MASP-1 and other inflammatory factors in the induction of proinflammatory changes in endothelial cells. As endothelial cells play a substantial role in the regulation of inflammation by balancing pro-and anti-inflammatory states, minor effects on endothelial cells can cause major inflammatory changes. Despite the use of suboptimal doses of all activators, we found several cases where Ca 2+ mobilization, the permeability of the endothelial layer, and the expression of cytokines and adhesion molecules were significantly enhanced by the treatments. These cooperations could also lower the threshold of endothelial cells to danger signals by enhancing the receptor expressions. In particular, we found considerable types of cooperation between MASP-1 and LPS, highlighting the risks of bacterial infections under conditions where the complement system was already activated or could be easily triggered.
All other reagents were purchased from Merck-Sigma-Aldrich (Darmstadt, Germany), unless otherwise stated.
Each experiment was performed in at least three independent, primary HUVEC cultures from different individuals before passage four. The study was conducted in conformity with the WMA Declaration of Helsinki; its protocol was approved by the Semmelweis University Institutional Review Board (permission number: TUKEB141/2015). All participants provided their written informed consent before inclusion.

Intracellular Ca 2+ Mobilization Assay
Intracellular Ca 2+ mobilization was measured using the method described previously [5]. HUVECs were seeded in 96-well plates in 100% confluency and cultured in Comp-MCDB medium for 24 h; then, the medium was changed to Comp-AIM-V for an additional 24 h. The cells were pretreated with the selected agonist in a suboptimal dose (rMASP-1: 0.6 µM, LPS: 10 ng/mL, histamine: 5 µM, IFNγ: 2 ng/mL, or bradykinin: 2 µM) for 24 h or not pretreated. An amount of 2 µM Fluo-4-AM (ThermoFisher Scientific, Waltham, MA, USA) was used to load the cells for 20 min; then, the cells were incubated in HBSS for another 20 min. Measurements were carried out with fluorescence microscopy; sequential images were taken every 5 s. To determine baseline fluorescence, two images were taken before adding the treatment. Twenty cells per image were analyzed using Olympus CellP 2.1 software.

Institutional Review Board Statement:
This study was carried out in accordance with the recommendations of the WMA Declaration of Helsinki, with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Semmelweis University Institutional Review Board (permission number: TUKEB141/2015).