Normal pressure wound healing
VEGFR expression in normal cells was highest for VEGFR1, followed by VEGFR2 and then VEGFR3, indicating that in healthy tissue (day 0). Furthermore, there was very little difference in the expression of the three receptors over time in terms of the overall degree of staining, whereas the occupancy results (proportion of all VEGFRs occupied by a given receptor) showed that the occupancy of VEGFR3 tended to be higher and that of VEGFR1 tended to be lower. From day 7 to day 14, the occupancy value for VEGFR3 increased further, and then the value of VEGFR2 increased thereafter. This result indicates that these upstream cascade reactions undergo changes during this time period. In particular, VEGFR3 is mainly activated by VEGF-C and -D related cascades, whereas VEGFR1 is mainly activated by VEGF-A, as well as VEGF-C and -D; thus, the branching point at days 7 to 14 appears to be represented by decreased activation of VEGF-A. Indeed, VEGF-A is a common activating factor of VEGFR1 and VEGFR2, which could explain the observed shift at day 7 and day 14, with the occupancy of VEGFR1 decreasing significantly on day 7 but that of VEGFR2 dropping to an extreme degree on day 14. In other words, the actions of VEGF-A on VEGFR1 and VEGFR2 predominantly affected the expression of VEGFR2 by day 7, with low occupancy of VEGFR1, but by day 14, the expression of VEGFR1 began to increase sequentially.
The main subsets of VEGF-A are VEGF-A121 and VEGF-A165, and their balance is relevant to VEGFR1 and VEGFR2 activity and expression. Mac Gabhann et al. [
24,
25] have stated that the action of VEGFR2 is intensified due to activation of VEGF-A165 in the presence of neuropilin, mainly in the process of muscle differentiation or in the vascular endothelium; in the absence of neuropilin, activation of both VEGF-A165 and VEGF-A125 resulted in more intense activity of VEGFR1 than VEGFR2. These facts suggest that on day 7 in this study, neuropilin was present in abundance in the tissue, and that its levels decreased by day 14. Furthermore, on day 28, VEGFR1 and VEGFR2 expression decreased significantly, but there was no change in the occupancy of VEGFR1 although that of VEGFR2 decreased. This implies that at the period from day 21 to day 28, VEGFR3 had also decreased; therefore, despite an overall change in the activity of the cascade in terms of VEGF-A, -C, and -D as a whole, there may have been a particular change in the balance of VEGFA in which VEGF-A165 might have decreased more than VEGFR-A125 in the absence of neuropilin.
The immunoreactivity of VEGFR3 peaked at day 21 and was significantly higher than the others, with the highest rate of increase. At day 14, there was a significant difference between VEGFR3 and VEGFR2 expression, which is therefore considered the time point representing a change in the VEGF-C and VEGF-D signaling cascades upstream of VEGFR2 and VEGFR3. It is unclear whether this difference is due to the more significant antigenicity of VEGFR3 compared to VEGFR2 or to the actions of VEGF-C and -D. VEGFR3 expression is indicative of differentiation of lymphatic vessel-like vascular epithelial tissues. However, the earlier increase in VEGFR3 on day 14 implies that the antigenicity of VEGFR1 and VEGFR2 was also elevated at this point.
In summary, considering previous findings in light of the present experimental results, we can conclude that under normal circumstances, lymphatic differentiation involves the complete cycle for VEGF-C: VEGFR3 is activated following VEGFR2 activation, and typical lymphatic differentiation takes place. The results of the present study also imply that changes in the antigenicity of VEGFR2 and VEGFR3 might ultimately converge to complete the formation of lymphatic vessels, especially from the aspects of recovery timing in wound healing.
NPWT
In the experimental group that underwent NPWT, the patterns of the emergence of expression of the VEGFRs were completely different from those in the control group. At day 7, the expression of VEGFR2 already reached its maximum value, followed by high expression of VEGFR3 and low immunoreactivity to anti-VEGFR1. In light of the results of the control, we assumed that activation of VEGF-C and -D had already initiated considerable differentiation of cells, particularly those exhibiting VEGFR2 expression at this time. Moreover, the fact that expression of VEGFR3 was significantly higher than that of VEGFR1 implies that VEGF-A activation had not yet begun. Given that NPWT induces VEGFR and accelerates wound healing, Kieesling et al. [
27] suggested that performing NPWT at the time of sternum closure after heart disease surgery would promote wound healing through vascular induction accompanied by cytokine induction. Furthermore, according to Bletsa et al. [
28], the binding of VEGF-A to VEGFR2 causes DLL4 induction in vascular tip cells, leading to Notch signaling in vascular endothelial cells to induce VEGFR2 and VEGFR3, thereby accelerating vascular endothelial cell and lymphatic vessel differentiation. Furthermore, VEGFR3, which is a principal receptor of VEGF-C, has been suggested to be more strongly induced by Notch [
26].
At days 14–21, all of the VEGFRs were active, and their expression levels were similar on day 21. As indicated above, VEGF-C and VEGF-D are considered common activation factors for all VEGFRs. The stable emergence of VEGFR3 was a particular characteristic feature of this period. Rissanen et al. [
29] and Anisimov et al. [
30] attempted to use adenovirus as a gene expression vector to induce VEGFR3 in response to external/long-term stress and found that VEGF-D, which acts on CAC base repetitions, likely has the higher contribution to stronger VEGFR3 promotion. In the present study, both VEGFR3 and VEGFR2 maintained high levels of expression continuously throughout the mid- and late stages (days 14–21) of NPWT, suggesting that NPWT induces VEGFR2 expression via a higher ratio of VEGF-C/-D than VEGF-A.
Yamanaka et al. [
31] stated that VEGFR2 promotion acts on the blood vessel and lymphatic systems and contributes to wound healing on the wound surface, and Bletsa et al. [
28] reported an association of VEGF-D with lymphatic repair ability. The high expression of VEGFR3 observed at the deep wound surface in the present experimental system was observed at days 14–21, with a constant intensity of tissue staining during this period, implying that VEGF-D-derived promotion factors would also be particularly relevant for the deep sites that were highly stainable.
Regarding lymphatic differentiation, our results suggested that differentiation of the blood vessel system was occurring as of day 14, owing to the rapid increase in the immunoreactivity and occupancy of VEGFR1. VEGFR1 expression peaked at day 21 and then declined rapidly on day 28. In contrast to the control group where VEGFR1 maintained its peak position, the peak at 21 days in the NPWT group was transient. Kumar et al. [
32] found considerable expression of VEGFR1, VEGFR2, and neuropilin-1 and -2 at the wound surface during postoperative repair from vascular system stress, and only VEGFR1 decreased rapidly after completion. In many aspects, this is consistent with the results of the present study, especially for the results of the control where, in the assumed presence of neuropilins, activation of both VEGF-A165 and VEGF-A125 likely allowed for VEGFR2 to be more readily expressed than VEGFR1, and therefore lowered the antigenicity of VEGFR1.
The fact that all VEGFRs showed abundant expression at day 21 is considered to be induced by the respective VEGF signaling cascades. In particular, the accumulation of VEGF-C exhibits this phenotype from the induction of the vascular system, which is represented by an increase in basic fibroblast growth factor (bFGF). Koolwijk et al. [
33] suggested that VEGFR3 is expressed in the lymphatic system, whereas VEGFR1 is expressed in the blood vessel system, and that their abundant and simultaneous expression causes induction of the fibroblast system. In particular, early expression or increased expression levels of VEGFR2 in the NPWT group are believed to help the functioning of fibroblasts that make up the blood vessel system and lymphatic system.
In light of the present experimental results, compared to normal wound healing, the NPWT group showed higher maximum activation values for VEGFR3 and VEGFR2, as well as earlier timing of maximum expression; however, in the case of VEGFR1, the maximum activation value was higher but the timing of maximum expression was the same as the control. Moreover, according to Joukov et al. [
34], expression of the Np2 gene and VEGFR3 results in an earlier transition of the timing of the generation of the vascular system in the embryonic stage via tyrosine kinase. In addition, the phenomenon of the earlier expression of VEGFR3 would require participation of a VEGFR3/Np2 activity system, which is a series of reaction systems [
35]. Ferrel et al. [
36] screened for genes involved in the Np2 activity system in humans and found that the upstream cascade involved in lymphatic regeneration in lymphoma was mainly activated by VEGF-C, which in turn activates VEGFR2 and VEGFR3 that share an Np2 activity system. This same phenomenon could be responsible for the expression patterns observed in the NPWT group, thereby producing an early increase in VEGFR3 based on Np2 as a growth factor of the lymphatic vessels and blood vessels.
In summary, with negative pressure applied to the wound surface, both VEGFR2 and VEGFR3 were expressed abundantly in the early stage in this experimental model, which induced early wound healing.
Potential mechanism and clinical prospects for NPWT
Erba et al. [
3] reported early induction of wound healing due to early high expression of the VEGF-C dimer and a urethane effect. Similarly, in the present experimental system, VEGFR2 levels were abnormally elevated in the NPWT group compared to normal wound healing group on day 7 (the first day of measurement). Incidentally, Erba et al. [
3] used a pressure of −125 mmHg, which is the same condition as the VAC treatment system used in our study. Therefore, in a VAC treatment system under similar conditions (−125 mmHg/continuous mode), these results strongly suggest that VEGFR2, VEGFR1, and VEGFR3 appear early and are involved in the differentiation and growth of the vascular system.
Plikaitis et al. [
1] found that NPWT was involved in myofibroblast construction after inducing granulation tissue to promote the creation of new blood vessel systems in the wound healing process. In the present experimental system, we immunohistochemically confirmed that expression of VEFGRs was activated on day 21 during normal wound healing, which was completed much earlier with NPWT, supporting the results of previous work.
To investigate the relevance of negative pressure force to wound healing, Zhou et al. [
2] created wounds on the backs of Göttingen minipigs and compared various wound-healing markers and the wound-healing area under various levels of negative pressure over time. Bacterial infection significantly decreased under all pressure levels, whereas capillary system regeneration and expression of the capillary proteins and VEGF and bFGF occurred at a relatively earlier stage in the low-pressure groups. The authors concluded that revascularization occurred earlier under NPWT at low pressure (−75 to −150 mmHg) based on the earlier activity of bFGF and VEGF, which promoted the formation and differentiation of granulation tissue.
Considering these previous findings and the results of the present study, the standard clinical use of a negative-pressure level of NPWT of −125 mmHg is regarded as valid. However, further research on different wound-healing states at even earlier stages after surgery (days 1–7) is required, and more detailed experimentation for each day would provide valuable insight into the mechanisms of NPWT, which could help to improve its clinical utility.
Finally, the specific focus of this experiment was to evaluate the reactions of VEGF-A, B, -C, and -D (VEGFR1, R2, and R3) using a rabbit model. However, in the wound-healing experiment, VEGF-F caused epidermal regeneration in NPWT in the same way as observed with VEGF-A to -D. In addition, the use of a porcine model would fundamentally allow for a comparison to human skin, since they have the same structure, and would further enable creation of a larger wound area. Given that one limitation of the study was that the mRNA expression level was not detected, further studies should be carried out using an in situ hybridization method. Thus, future work should focus on understanding the wound-healing mechanism of NPWT using VEGFR and a porcine model, which will likely help in obtaining clearer and more clinically translatable results.