Analysis of molecular mechanisms of regenerative processes in tissues of patients with diabetic foot syndrome

Introduction

Currently, diabetes mellitus (DM) is a pressing healthcare problem worldwide. According to literary sources, the number of people with DM has increased by more than 2 times in the last few years. It should be noted that in the Russian Federation, there has also been a significant increase in people suffering from DM [1].

It is known that one of the late DM complications can be diabetic foot syndrome (DFS) which is infection, ulcer, or destruction of foot tissues associated with neuropathy and/or decreased main blood flow in the arteries of the lower extremities [2, 3], which in turn is accompanied by a violation of the tissue regeneration process. Changes occur in all phases of wound healing: hemostasis/coagulation, inflammation, proliferation, epithelialization, and scar formation [4], which is accompanied by an increase in healing time and the formation of chronic wounds/ulcers. As statistical research data demonstrate, DFS was recorded in 5.6% of patients with T1DM and in 2.4% of patients with T2DM [2]. However, according to some research, the mortality rate in patients with DM who developed non-healing ulcers over five years reached 40% [3].

According to statistical research, mortality after amputation varied from 13% to 40% one year after surgery, from 35% to 65% — three years after surgery, and from 39% to 80% — five years after surgery [5, 6, 7]. Thus, this pathology is a serious challenge for researchers and healthcare worldwide.

This study was aimed at analyzing various biochemical mechanisms involved in the process of wound regeneration in patients with DFS.

Materials and methods

This study presents an analysis of literature data on the biochemical mechanisms involved in wound regeneration in patients with DFS. In order to achieve this goal, articles were selected and analyzed in the foreign databases PubMed, MedLine, and Google Scholar, as well as in the Russian Science Citation Index (RSCI) database for the period from 2017 to 2023. The search for articles was conducted using the keywords diabetic foot, wound healing, molecular mechanisms and their analogues in Russian, such as «диабетическая стопа» (diabetic foot), «заживление ран» (wound healing), «молекулярные механизмы» (molecular mechanisms). The search was conducted using words in titles, abstracts, and texts. The selection of articles was performed using publication dates from 2017 to 2023 by article type (systematic reviews, meta-analyses, randomized controlled trials, clinical trials, and availability of full text in the public domain). Articles published before 2017 and those not relevant to the topic of the study were excluded. During the literature search, 74 publications were identified, of which 24 literary sources published from 2017 to 2023 and corresponding to the direction and purpose of the study were included in the literature review, and additional 18 articles published later than 2017 and being necessary to disclose the subject of the study from the references in the bibliography were used.

Results

The performed literature data analysis on the study of molecular mechanisms of regenerative processes in tissues in patients with DFS allowed identifying the main issues characterizing the peculiarity of the healing processes in this category of patients.

The healthy human skin barrier depends on a well-regulated balance of lipids, intercellular junctions, antimicrobial peptides, and enzymes preventing water loss and infection [7]. The outermost layer of the skin (stratum corneum, i.e. horny layer) is composed of terminally differentiated, anucleate keratinocytes filled with keratin fibers and associated proteins called corneocytes, which are surrounded by a hydrophobic lipid layer and protect against transepidermal water loss (TEWL) [7]. While aging, the skin naturally experiences decreased lamellar body secretion, depleted lipids, slower barrier repair, and increased TEWL [7]. Although the total water content of the stratum corneum decreases with age [8], the water content of the superficial stratum corneum is similar in both young and aged skin [9]. It has been established that the skin of patients with DM has a reduced lipid content and reduced hydration of the stratum corneum, although some studies did not note significant changes in TEWL [10], while other literature sources indicated an increase in TEWL [11]. A study conducted in 2017 revealed changes in TEWL in patients with DM [12]. The research data of some authors suggest that skin hydration apparently correlates with microcirculation [13] and is a significant predictor of wound healing [14].

DM-associated hyperglycemia leads to protein glycosylation and changes in epidermal structure, which is accompanied by disruption of the ultrastructure of basal cells and, as a consequence, disruption of the normal function of the skin barrier [15], further increasing the risk of wound infection. Some authors note that the skin of patients with DM has higher colonization of S. aureus and epidermal strains of bacteria, while common bacterial colonizers such as Staphylococcus, Pseudomonas, and Enterobacteriaceae have been identified in patients with diabetic foot ulcers [16]. Bacteria such as staphylococci and streptococci produce proteolytic factors that destroy the skin barrier [17].

Researchers have identified that the pH in the wound surface area of ​​diabetic foot is significantly more alkaline compared to normal wounds [11]. Alkaline environmental conditions promote biofilm formation, which differs from healthy skin, and also affects the resistance of bacteria to antibiotics [18]. Thus, when prescribing treatment for an infection detected in the abnormally alkaline environment of the diabetic foot, this difference in the sensitivity of bacteria to antibacterial agents should be considered [7][19].

Under hyperglycemic conditions, the polyol pathway of glucose metabolism (conversion of glucose to sorbitol with subsequent formation of fructose) is excessively activated, which leads to a decrease in the concentration of nicotinamide adenine dinucleotide phosphate (NADPH) [20]. This ultimately disrupts the functioning of aerobic mechanisms (production of reactive oxygen species (ROS) and respiratory release), which are involved in the process of microbial inactivation by neutrophilic granulocytes [21]. Relative insulin deficiency associated with insulin resistance in T2DM or its complete absence in T1DM type 1 leads to a decrease in the activity of insulin-dependent enzymes in neutrophilic granulocytes and also disrupts their migration. The processes such as chemotaxis and phagocytosis that depend on monocytes and macrophages are also disrupted [20].

Under normal functioning conditions, macrophages secrete cytokines and growth factors necessary for the wound healing process [22]. However, under hyperglycemia, the transition of the proinflammatory phenotype M1 of macrophages to the anti-inflammatory M2 is disrupted [23]. There is evidence that tumor necrosis factor-α (TNF-α) and interleukin (IL)-1β participate in the control of this process, preventing the transition to the anti-inflammatory M2-type macrophages [23] and regulating the inflammatory response and tissue regeneration. As the scientific literature review shows, there is a relationship between the level of insulin in the human body and the functional state of leukocytes. In patients with DM, a decrease in insulin levels can lead to dysfunction of leukocytes and, as a consequence, to an immunodeficiency state [25], which complicates the process of tissue regeneration.

Patients with diabetes have increased levels of inflammatory cells, but this is not accompanied by an improved immune response. There is evidence that chronic wounds contain more B lymphocytes, plasma cells, and T cells with a low CD4+/CD8+ ratio [23].

In the proliferative phase of healing in DM, which includes three main components (fibroplasia, angiogenesis, and epithelialization), all stages of healing are accompanied by changes. Herewith, changes in the physiology of fibroblasts and their resistance to the action of IGF-1 and EGF are observed [23][26]. According to research data, in response to ischemia/hypoxia in the lower extremities of patients with DM, a decrease in the migration and proliferation of fibroblasts is observed [27]. Also, with hyperglycemia, the phenotype of fibroblasts changes to "prodegradative", which leads to a decrease in the production of normally functioning collagen, an increase in the secretion of metalloproteases (MMPs), and also a decrease in the synthesis of nitric oxide (NO) [23]. Numerous studies in various regions have reliably indicated that under conditions of a persistent and prolonged increase in glucose concentration in blood, collagen glycation occurs resulting in the significant reduction of fully functional collagen formation in wounds despite the high expression of type I collagen genes [27].

Endothelial dysfunction (ED) also plays an important role in tissue metabolism disorders in DM [28] and is one of the causes of tissue regeneration disorders. ED is associated with impaired vascular wall reactivity, which leads to impaired blood flow in the distal sections and the formation of arteriovenous anastomoses [21]. Research data demonstrated that in some patients with DM, impaired neurohumoral regulation was manifested by persistent arteriospasm and, as a consequence, orthostatic distal angiohypertension causing the development of distal angiitis, which led to local ischemic disorders in the lower extremities [29].

Hyperglycemia influences ED development through several pathways, including the polyol, hexosamine, advanced glycation end products (AGEs), and protein kinase C (PKC) pathways [21]. The polyol pathway, which metabolizes glucose, leads to ED by reducing NO supply to dysfunctional vessels due to a decrease in cytosolic NADPH. A decrease in the concentration of NADPH, which is necessary for the regeneration of glutathione, as well as for the production of NO by the enzyme NO synthase, leads to persistent vasoconstriction and disruption of antioxidant systems [21]. The PKC pathway affects directly the regulation of endothelial vessels. An increase in the procoagulant activity of endothelial cells is achieved by reducing NO formation and accelerating vascular cell proliferation [30]. As a result, a change in the priority of metabolic pathways for glucose utilization induces the progression of ED.

In the tissues of patients with DM, as a result of metabolic transformations of glucose, the concentration of triose phosphates increases: 3-phosphoglyceraldehyde and dihydroxyacetone phosphate. Dihydroxyacetone phosphate serves as a substrate from which methylglyoxal is formed [31–34]. The presence of hyperglycemia activates the process of non-enzymatic glycosylation of proteins [35], and the presence of the linear form of glucose leads to the formation of Schiff bases. They serve as a substrate for the formation of Amadori products, which are converted into end products of non-enzymatic glycation (AGEs) [36] as well as low-molecular dicarbonyls [31] which include glyoxal and methylglyoxal. Similar to malondialdehyde, its homologue glyoxal and structural isomer methylglyoxal cause tissue damage, which leads to disruption of the tissue regeneration process [37].

Hypoxia of lower limb tissues leads to intensification of the lipid peroxidation (LPO) process, which is controlled by the antioxidant defense system [38]. Patients experience uncontrolled activation of LPO processes, which is accompanied by the release of tissue decay products into the systemic bloodstream, which leads to the development of mutual aggravation syndrome [38]. Malondialdehyde, being a secondary intermediate product of LPO, as a highly active aggressive compound non-enzymatically forms protein-protein, lipid-lipid, and protein-lipid bonds (Schiff base), thereby disrupting the transport function of various cell membranes and the function of biologically active molecules [37]. The formation of ROS, intermediate and final LPO products aggravates the dysfunction of endothelial cells, thereby mediating DNA damage [21]. Increased activity of the hexosamine pathway, which occurs due to hyperglycemia, leads to the formation of uridine phosphate-N-acetylglucosamine, which glycosylates proteins [21]. An increase in the concentration of uridine phosphate-N-acetylglucosamine leads to increased expression of TGF-β1 and plasminogen activator inhibitor-1 (PAI-1) [21], which contributes to the formation of microclots and the development of endotheliopathy.

In patients with DM, metabolic changes induce impaired regulation of MMPs, which enable endothelial cells to migrate to the site of injury [23]. Multiple growth factors are involved in controlling the activity of metalloproteases and tissue inhibitors of MMPs (TIMPs), including TGF-β1, IGF-1, FGF, IL-10, EGF, and monocyte chemotactic protein-1 (MCP-1) [39]. TGF-β1 usually blocks the expression of genes encoding various types of MMPs, but this does not occur in DFS [23]. Thus, MMP9 can cleave fibronectin into fragments, which enhances the migration and proliferation of inflammatory cells and prolongs the inflammatory process [40], and the ratio of angiopoietins in the wound simultaneously changes [41]. The above-mentioned changes induce disruption of the stages of vessel formation in tissues in patients with DFS [41].

It is important to note that hyperglycemia leads to a reduced ability of cells to adapt to hypoxic conditions due to a decrease in the expression of hypoxia inducible factor-1 (HIF-1) [23], which in the body is represented by two heterodimers: HIF-1α and HIF-1β. It is the decrease in HIF-1α, regulated by IGF-1 [42], that disrupts the regulation of the expression of many factors involved in the process of migration, proliferation, and angiogenesis. As a result, before the onset of tissue ischemia in the lower extremities of patients with DM, hyperglycemia creates unfavorable conditions that reduce cell survival and prevent adaptation processes [23].

Discussion

An increase in the number of patients with DM induces an increase in the need for wound treatment in DFS which requires further research into the molecular mechanisms due to the fact that some patients experience postoperative suture failure despite insulin therapy.

The obtained research data on the skin condition in patients with DM indicate a decrease in water content, which is associated with impaired microcirculation. A decrease in the amount of water in the tissue leads to thinning of the hydration shell of channel proteins, various ions, biologically active molecules, and cellular receptors, which disrupts their functional activity. Obviously, receptors of a protein nature that have lost their functional activity are not able to interact with biologically active molecules, in particular with insulin. Since the connective tissue is an insulin-dependent tissue, it can be supposed that receptors that have lost their functional activity, against the background of insulin therapy, are not able to respond to biologically active molecules, which affects the decrease in metabolic processes in the tissue and the rate of collagen synthesis. Changes in the functional activity of channel proteins that have lost the required thickness of the hydration shell also lead to a disruption of their functional activity. The consequence of the above processes is a decrease in the work of cellular transport systems. Fewer ions, glucose, and amino acids enter the cell leading to a decrease in energy metabolism, which in turn disrupts the regenerative capacity of the tissue.

It should be considered that in a patient with DFS, due to the severity of the dysfunction of the immune system, the wound healing time may increase, which increases the risk of infection and may subsequently lead to open wound management.

A large number of studies indicate that the development of orthostatic distal vascular hypertension in patients with DM is due to changes in the priority of metabolic pathways for glucose utilization, which is one of the reasons for ED development [21][28-30]. It is necessary to consider the fact that under conditions of hypoxia development, LPO processes are activated, which are not fully controlled by the antioxidant defense system [21][37][38]. It should be mentioned that adaptive cell responses in patients with DM under conditions of hyperglycemia are reduced due to a decrease in the expression of HIF-1α [23][42]. All these changes are accompanied by the development of mutual aggravation syndrome [38].

The above factors form a "vicious circle", mutually reinforcing each other's action, which can contribute to the disruption of regenerative processes in the patient's body. Analysis of the biochemical mechanisms of these data expands the understanding of pathological issues leading to complications of postoperative wounds. Such studies will allow targeted action on the wound healing process by means of specialized corrective methods taking into account individual characteristics, as well as improve the quality of life of patients.

Conclusion

The conducted literature data analysis has demonstrated that regenerative mechanisms in patients with DM include a complex chain of biochemical reactions that depend on the state of the skin barrier, the functional activity of immunocompetent cells, the number and ratio of biologically active molecules, as well as the ratio of various classes of lipids, intercellular compounds, antimicrobial peptides, enzymes and the degree of severity of angiopathy.

Considering the peculiarities of tissue metabolism in patients with DFS, which can often be complicated by surgical suture failure, a personalized approach to pharmacological therapy is required. Before surgery, such patients should be recommended drugs that improve tissue microcirculation, as well as recommended the use of antioxidant therapy to reduce the destructive LPO processes. In the preoperative period, skin microflora should be tested for aerobic, facultative-anaerobic flora with determination of sensitivity to antibiotics and selection of the minimum inhibitory concentration of the drug to increase the efficiency of the treatment in the postoperative period. For a more complete understanding of the mechanisms of regenerative processes at the molecular level, it is advisable to search for new factors involved in the mechanisms of DFS development to prevent complications and increase the efficiency of the treatment.