DFUs are often challenging to treat, and these wounds can become stagnant even if the best available treatment is provided. Previous research has shown that medications primarily used to control blood sugar levels, such as DPP4i(s), may help heal DFUs [12]. Consistent with these findings, our last study of diabetic patients with DFU found that DPP4i Vildagliptin improved healing by approximately 35% compared to placebo [6]. Therefore, we investigated the molecular mechanisms by which Vildagliptin might promote wound healing.

Diabetes mellitus may lead to various microangiopathies, including DFUs, often due to undergoing endothelial cell (EC) dysfunction [13, 14]. Interestingly, DPP-4i(s), including Vildagliptin, was found to protect from EC dysfunction [15]. Perhaps, even more importantly, the protective effect is seen even in a normoglycemic context where Vildagliptin attenuates EC dysfunction in a non-diabetic mouse model [16]. This significant finding suggests that Vildagliptin may also affect cellular processes beyond its canonical inhibition of DPP-4. To further elucidate these findings, we assessed the differential expression of selected genes critically involved in essential cellular functions, some of which were evaluated previously and sometimes in different contexts. For example, Pujadas and colleagues previously reported that another DPP4i, Teneligliptin, increases the proliferation of human umbilical vein endothelial cells (HUVEC) exposed to hyperglycemia [17]. However, we did not find any effect of DPP-4i Vildagliptin on levels of cell proliferation markers in wound fluid obtained from diabetic patients with DFU. Likewise, it is indicative that DPP4i(s) might attenuate EC senescence in vitro and animal models [18, 19]. Still, we did not find a similar effect of Vildagliptin on the expression of senescence markers in DFU wound fluid in our patients.

Nevertheless, Zhao and colleagues reported that the DPP4 enzyme promotes EC apoptosis and autophagy [20]. Although vildagliptin did not affect the expression of apoptotic markers in our context, our findings suggest that it attenuates autophagy. This is consistent with a study by Zhao and colleagues [20]. Our results showed that Vildagliptin treatment for 12 weeks resulted in a more than twofold reduction in the mRNA levels of the autophagy marker nucleoporin 62 (NUP62 or p62) in the DFU wound fluid. Autophagy is an essential physiological cell self-renewal process; however, if in excess, it can trigger so-called autophagic cell death due to excessive degradation of cellular content [21, 22]. The effect of vildagliptin on NUP62/p62 mRNA levels may be due to its ability to control blood sugar levels, which indirectly affects autophagy. When cells are exposed to a high concentration of glucose, the level of O-linked N-acetylglucosamine (O-GlcNAc) modification of the p62 nucleoporin increases [23]. Nucleoporins, including p62, are constitutively O-GlcNAcylated [24]. These modifications protect them from ubiquitination, thus proteasomal degradation [24], a hallmark of autophagy [9]. Indeed, p62 is a receptor for intracellular cargo to be degraded by autophagy, including ubiquitinated proteins [25, 26]. Hence, p62 is used as an autophagy marker.

An intriguing and somewhat unexpected finding emerged regarding the necroptosis marker RIPK3. After twelve weeks, RIPK3 levels significantly decreased in the wound fluid of DFU patients from both the Vildagliptin and placebo arms. Necroptosis is often seen as harmful to wound healing because of its pro-inflammatory nature [27]. However, the decrease in a necroptosis marker in both groups, although not completely understood, may suggest reduced inflammation within DFU, possibly creating a more favorable environment for healing. It is worth noting that there was a slightly higher rate of DFU improvement in the Vildagliptin group (8 out of 8 patients) compared to the placebo group (6 out of 9 patients) at the end of the study. This finding warrants further investigation into the interplay between RIPK3 and Vildagliptin’s mechanism of action in wound healing.

Furthermore, this study establishes the utility of filter paper absorption for collecting wound fluid samples to monitor multiple healing biomarkers within DFU. This minimally invasive method provides biological material for detecting local changes that might not be reflected in the circulation [6].

Our study has limitations. Due to the primary clinical use of DFU wound fluid, we could only collect samples from a relatively small group of patients (N = 17) who completed the 12-week treatment. While filter paper absorption is a patient-friendly method, it yields a lower sample volume than aspiration techniques. This limited volume necessitated qPCR, a highly sensitive method ideal for small samples. However, this approach focuses on gene expression and may not capture protein levels exactly. Despite these limitations, we could comprehensively evaluate 18 genes related to six critical cellular processes. Future studies with larger sample sizes could be more focused now and further substantiate our findings by incorporating protein testing methods. Second, given the filter paper absorption sampling, we expected more subtle differences in molecular marker expression within the collected samples because the DFUs had only partially closed by week 12. Finally, it is essential to note that wound fluid composition is complex and includes genetic material from various resident cell types, not just endothelial cells. Despite these limitations, our data provide valuable molecular insights into the ongoing processes within DFUs during the systemic administration of Vildagliptin.