VM is a recently discovered angiogenetic process found in many malignant tumors in which vessels are formed independently of vascular endothelium as in the typical angiogenic process. In contrast, it involves the formation of vascular structures composed of tumor cells, which generate a channel network facilitating blood supply for tumor growth [54, 55]. Many studies have pointed out that some clinical anti-angiogenic treatments have not been satisfactory, which could be, in part, attributed to VM processes. VM is therefore associated with poor prognosis, reduced survival and high risk of cancer recurrence in addition to resistance to anti-angiogenic treatment [13, 54, 56]. This process has been documented in many different human tumors [57], but to our knowledge has never been reported in normal physiological blood vessel formation. Therefore, in this study we aimed to investigate whether a process closely resembling VM can also occur during the regeneration of normal tissue. In addition, we wanted to identify the main cell type involved in this process. To address this question, the well-described zebrafish fin regeneration assay [34] was employed.

It has been previously described that a functional microcirculation network in malignant tumors is built by three patterns: (i) pre-existing endothelium-dependent blood vessels; (ii) mosaic blood vessels; and (iii) VM channels. Pre-existing endothelium-dependent blood vessels are incorporated into the tumor lesion, mosaic vessels are lined by both tumor cells and ECs, and VM channels contain tumor cells that line channels and mimic endothelial function [12, 58, 59]. Similar microcirculation networks have been documented for the first time in zebrafish caudal fin regeneration at 3dpa containing three vascular structures: normal blood vessels built by well-differentiated ECs, mosaic vessel containing typical EC and Endothelial-Like Cells (ELC), and channels built by ELCs (Fig. 1). This vascular network is fully perfused, as indicated by the presence of red blood cells (RBC) in the lumen. Rounded ELCs extend long sleeves surrounding vessel lumen and morphologically, ELCs closely resemble the macrophage (MΦ) structural phenotype. At the vascular front, multiple extravascular RBCs surrounded by multiple MΦs are revealed (Fig. 1a). Furthermore, we detected perfused mosaic blood vessels built by well-defined ECs and atypical cells appearing as channels in the regenerating fin at 7dpa (Fig. 2). At the channel tip, this cell appears to be a MΦ that makes contacts with the ECs. This cell extends cytoplasmatic extensions and elongates into the blood vessel. Based on this morphological observation, we then examined the MΦ appearance in vivo in the regenerating fin at the 7dpa. MΦs appeared near blood vessels, in the interray fin region, and in the front of the vessel tips (Fig. 2, Video 1). MΦs in front of vessel tips are actively interacting with ECs; making contacts and moving between them (Video 1). It is possible that they are involved in the tissue remodeling process by making channels and providing (drilling) space for the vessel growth. Similar roles of MΦs have been documented in breast cancer, where Obeid and colleagues [60] showed that tumor-associated macrophages (TAMs) could function as channels helping tumor cells to invade the ECM and thus contribute to tumor growth, and metastasis through an angiogenic pathway. Later in 2016, Barnett at al. reported a new role for TAMs forming functional VM channels proximal to cancer stem cells in melanoma [43]. In addition, an association between MΦs and blood vessel sprouting has been shown in vitro and in vivo during embryonic development [42], but MΦs obtaining an ELC phenotype and making perfused channels closely resembling the VM process, to our knowledge, has not been reported in normal regenerating tissue.

During the VM process, aggressive tumor cells can transdifferentiate into multiple cellular phenotypes and obtain endothelial-like characteristics and form channels that mimic blood vessel function [11, 61]. We therefore investigated whether MΦs possess a similar role to tumor cells during VM in the regeneration process of normal tissue. To examine phenotypic changes, flow cytometry analysis and immunofluorescence staining were performed revealing the presence of cells expressing both EC (fli1a) and MΦ (mpeg1) markers, suggesting that these cells are possibly differentiating from one cell type (MΦ) into other (EC) (Fig. 4). Both markers (fli1a and mpeg1) are typically cell line-specific and not co-expressed in the same cell line. It is well know that zebrafish fli1a labels EC and is highly expressed in vascular endothelium [62]. Therefore, the transgenic line is frequently utilized to observe individual migrating ECs [63]. On the other hand, mpeg1 was identified as a gene with expression tightly restricted to macrophages and has subsequently been used as a marker for this cell lineage in zebrafish [47]. According to the flow cytometry study, the percentage of double positive cells over 10 days has a Gaussian distribution in which the peak is reached by 3dpa. Interestingly, the peak is reached at the same time point in which multiple ELC and mosaic vessels were documented (Fig. 1). Afterwards, the amount of double positive cells (in %) decreased. We believe this is the result of cells differentiating into ELCs and adopting an EC phenotype, as documented by morphological observation (Fig. 2). Furthermore, double positive cells were confirmed by IF staining, where EC and MΦ markers were detected within one cell (Fig. 4).

To elucidate whether the process closely resembling VM is present during the regeneration of normal tissue, the VM inhibitor, CVM-1118, was used. Previous studies in vitro have reported that CVM-1118 results in reduced proliferation, induced apoptosis and importantly, reduced formation of branching, tubular networks that are characteristic of VM [10]. Our data, in vivo, demonstrated severe impairments in tissue regeneration and blood vessel formation described by a shorter vascular plexus, less pronounced blood vessels, and the absence of the regeneration process, including blood vessel formation, in half of the fin area (Fig. 5). In the fin area where the regeneration process is present, tissue and vascular plexus appear shorter with dense capillary meshwork containing multiple sprouts, indicating that regeneration is most likely supported by sprouting angiogenesis and not by VM. Interestingly, TRA and VPA are decreased, which means that the fin regeneration and blood vessel formation is impaired (Fig. 5). The reason for the complete absence of half of the regenerating fin could be due to induced apoptosis, reduced proliferation and severe disruption of the formation of vascular networks caused by CVM-1118, which is consistent with previous in vitro studies [10]. In addition, our data showed that CVM-1118 has a cytotoxic effect on MΦs, specifically, significantly reducing their numbers by 60% in comparison with the control animals (Fig. 6). To our knowledge, this effect on MΦs is documented for the first time and further studies involving molecular pathways underlying the interaction between CVM-1118 and MΦs are needed. Taken together, treatment with the vascular mimicry inhibitor (CVM-1118) significantly impairs tissue regeneration by obstructing vessel formation and expansion. Additionally, cytotoxic effect on MΦs has been show.

Further, we decided to investigate the effect of macrophage inhibitor PLX-3397 on caudal fin regeneration, blood vessel formation and MΦ appearance. It is well known that PLX-3397 inhibits the survival, differentiation, and proliferation of MΦs [50, 64, 65]. The role of the PLX-3397, its target, colony-stimulating factor 1 receptor (CSF1R), and impact on macrophages has been mainly investigated in the tumor environment. It is known that CSF1 plays a significant role in the recruitment of peripheral blood monocytes to the TME, differentiation into macrophages, and polarization of macrophages toward an M2-like phenotype via binding to the CSF1 receptor. It has been demonstrated that PLX3397 suppresses survival, migration, and M2 polarization of sarcoma TAMs, and induces their depletion [66,67,68]. Additionally, it has been shown by Conedera and colleagues in 2019 [50] that PLX3397 treatment significantly diminishes the influence of microglia-macrophages on the injury response. In our study, PLX-3397 was used to reduce the amount of macrophages present after the fin amputation (Fig. 6). Consistent with previous studies, our data showed a significantl reduction in MΦs (about 50%) upon treatment relative to controls. Moreover, the regenerative area and vascular plexus are impaired, capillaries are less prominent and the intraray region is improperly vascularized (Fig. 7), indicating that MΦs play a significant role in supporting proper tissue regeneration and vascularization. Furthermore, In vivo observation are supported by quantification using TRA, VPA and MΦ quantity, showing that upon PLX-3397 treatment all three variables are significantly decreased. Besides well-known importance of MΦs in proper fin regeneration [39, 40], we show the importance of MΦs in proper blood vessel formation during caudal fin regeneration where they contribute to the process closely resembling VM. Along with this, we think that by applying the MΦ inhibitor PLX-3397, we are reducing the amount of MΦs, which will subsequently transform in ELCs and help to form functional blood vessels.

The relationship between MΦs and tissue regeneration has been well studied and documented, as MΦs were shown to be essential during wound healing, tissue repair and tissue remodeling [69, 70]. MΦs display a high-functional plasticity with respect to shape, behavior changes during the immune response, gene expression, and supporting sprouting angiogenesis by bridging the gap between ECs in damaged endothelium and secreting some angiogenic growth factors [41, 42, 71, 72]. Here, we have revealed an additional characteristic of MΦs; demonstrating their capacity to differentiate into ELCs, adopt EC morphological features and contribute to neoangiogenesis by VM in the regenerating tissue. This opens new questions and gives a fresh perspective on the VM process.

In the present study, we are able to show that a process closely resembling VM, is present as well during the regeneration of normal, healthy tissue. During the early tissue regeneration process, vasculature is built by the mosaic blood vessels containing both ECs and ELCs. ELCs appear, morphologically, like MΦs and express both EC and MΦ markers (detected as double positive cells). Those cells appear at the regenerating front, adopt an EC phenotype and form functional and perfused channels. The vasculature is therefore partially expanded by transformation of the adjacent MΦ into ELCs. The mechanisms behind this process, however, still require further investigation. Overall, our study opens new perspectives on the physiological role of VM contributing to blood vessel regrowth during tissue regeneration and opens a window for innovative therapeutic options.