Correlational analysis of the regulatory interplay between molecules and cellular components mediating angiogenesis in wound healing under normal and hyperglycemic conditions

Abstract

AIM:

The aim of this study was to correlate the content of cells with regulatory molecules associated with angiogenesis in wound healing in a rat model of hyperglycemia. We hypothesize that blood neutrophils are the main VEGF source and can stimulate FLT-1 receptor expression, which is the perquisite for efficient neoangiogenesis.

MATERIALS AND METHODS:

Kinetic studies of the healing dynamics (3, 7, 14, 21 days) of burn wounds on the skin were conducted in white adult male rats. The content of nuclear factor kappa B (NF-κB), vascular endothelial growth factor (VEGF), its receptor (Flt-1) in the regenerated tissue was analyzed by western blot. Numbers of cells associated with the regenerative process and from peripheral blood (PB) were determined. Additionally a bone marrow (BM) myelogram was conducted.

RESULTS:

The relative number of peripheral blood (PB) neutrophils was found to be associated with the level of VEGF (R = 0.708) and Flt-1 (R = 0.472). The relative number of fibroblasts was also associated with VEGF (R = 0.562), but not with Flt-1. A negative association was found between the number of neutrophils in the regenerated tissue with VEGF (R = –0.454) and FLT-1 (R = –0.665). This confirms our hypothesis, that blood neutrophils are the main VEGF producer that stimulate the expression of the FLT-1 receptor subsequently inducing neoangiogenesis.

Furthermore, that under hyperglycemic conditions fibroblasts were highly associated with VEGF (R = 0.800), while negatively associated with FLT-1 (R = –0.506). There was a high association between PB neutrophils and newly generated tissue cells: neutrophils (R = 0.717) and macrophages (R = 0.622), as well as the association between neutrophils and macrophages (R = 0.798). This is an indication of chronic inflammation and increased transmigration of blood cells to the burned tissue.

CONCLUSION:

Blood neutrophils are the main producer of VEGF and stimulate the expression of the FLT-1 receptor. In the context of hyperglycemia the imbalance of receptor and ligand associated with angiogenesis indicates for chronic inflammation: VEGF and FLT-1, which facilitates hypoxia, prevents the physiological course of burn wound healing and may be an important factor in impaired tissue regeneration in diabetes.

Keywords

Angiogenesis granulocytes neutrophils macrophages connective tissue burn wound

1 Introduction

The interaction of cellular reactions in connective tissue is the basis for successful wound healing. Inflammation, regeneration, and fibrosis are essential components of the tissue’s response to damage. This reaction is mediated by the interaction of blood cells such as neutrophils, platelets, macrophages, and lymphocytes, with cells of the connective tissue including fibroblasts as well as feedback mechanism mediating the rearrangement of extracellular matrix. The role of the “conductor in the cell orchestra” changes at different stages of the regenerative process. Macrophage-fibroblastic interaction is most important in the association between inflammation, regeneration and extracellular matrix formation [1, 2]. The dysfunction of autoregulatory mechanisms in particular, intercellular interactions can lead to prolongation of inflammatory reactions, incomplete phagocytosis, impaired regeneration, progressive sclerosis, the formation of pathological granulation and fibrous tissue or chronic open wounds [2 –4].

It has been established, that in the case of carbohydrate metabolism disorders, the healing process of wound defects can be slowed down both by decreased levels of local growth factors and due to the presence of micro- and macrovascular impairments [7, 8]. Decreased peripheral sensitivity, local hemomicrocirculation disorders, chronic hyperglycemia increase the risk of trophic ulcers and potential infections [7, 9].

Angiogenesis is an important part of the repair process. This is a dynamic process, which is regulated by both systemic signals from blood compartments and locally from the extracellular matrix. Vascular endothelial growth factor (VEGF), angiopoietin, fibroblast growth factor (FGF) and transforming growth factor beta (TGFβ) are the most potent angiogenic mediators [10, 11]. Thus, VEGF, whose expression is controlled by NF-κB, is a key transcriptional regulator of blood vessel formation and mediates chemotactic effect on endothelial cells [12]. Both human and animal neutrophils have been shown to be a source of VEGF [13]. Tissue infiltrating neutrophils produce not only VEGF, but are able to interact with epithelial cells, inducing the production of VEGF in them [13 –15]

However, it remains debatable which cells are the main VEGF producers, and which cells stimulate the production of VEGF subsequently regulating the tissue regeneration. This question is important because the deficiency of trophic factor in the connective tissue is the main reason for the decrease in angiogenesis in the tissues of hyperglycemic patients [16 –18].

The main purpose of the study was to explore possible correlations between the content of newly generated tissue cells and regulatory molecules that can facilitate angiogenesis during wound healing under hyperglycemic conditions.

2 Materials and methods

All animal experiments were carried out in compliance with generally accepted bioethical norms of humane treatment of laboratory animals in accordance with international and national provisions on conducting experiments involving animals: “European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes” (Strasbourg, 1986); “General ethical principles for conducting experiments on animals” (Ukraine, 2001), the Law of Ukraine “Animal protection from cruel treatment” No. 3447-IV (Ukraine, 2006), meeting of the Bioethics Commission in NMU, Protocol N94 of 03/16/2016.

The healing dynamics (on the 3rd, 7th, 14th, 21st day) of the skin burn wound was explored using mature male rats Wistar (176,8±8,3 g) without somatic pathology i. e. “control group” (n = 24) and on rats (n = 24) with experimentally induced diabetes. Diabetes mellitus was induced by the intravenous administration of streptozotocin in a single dose 50 mg/kg. To describe the data in the groups, the values of the median (Me) and percentiles of the 25th (P25) and 75th (P75) were given, which were determined [QI ÷ QIII]. Evidence of the development of hyperglycemia after 1 month was a glucose level of 12.35 [11.2 ÷ 16.68] (10.2–19.00) mmol/l against control values of 5.37 [4.7 ÷ 5.9] [4, 2–6.6) mmol/l. The level of HbA1 was 7.8 6.9 ÷ 10.2] (6.9–12.2) % versus 4.4 [3.8 ÷ 4.9] (3.6–5.2) % in the control.

2.1 Animal experiments

We exposed a rectangular area on the trunk skin applying depilation cream for 4-5 minutes in a free rat behavior some days before to perform the experimental model of the burn. After the procedure we treated the skin with saline and antiseptic solutions. The defined area was not damaged, evenly smooth, without hair. The next day, the skin part of rats under the influence of ether anesthesia were burned. Two copper plates were used in the elliptical shape, 3.7×4.5 cm in size and 0.0052 m² square. The plates were kept in boiling water (100°C) for 10 minutes and were applied simultaneously symmetrically on both exposed parts of the rat body for 10 seconds. The area of thermal skin damage was deducted from the rat body surface. The total area of the skin burn was 18–20% of the body, which is sufficient for the formation of the dermal superficial burn on the II degree. At the indicated time (on 3rd, 7th, 14th, 21st day), we injected a lethal dose of sodium thiopental intraperitoneally and sacrificed the rats.

2.2 Bone marrow analysis

A myelogram study and the calculation of the peripheral blood elements were carried out by a visual assessment of the blood smear and using a micro-hematology analyzer MicroCC. To obtain the bone marrow, we separated the rat femur from the muscle tissue. The bone marrow material was extracted from the distal epiphysis of the femur onto a defatted glass slide. Prepared smears were fixed with ethanol and stained by Romanovsky-Giemsa. On each glass, 500 cellular elements were counted, the number of cells of each type was determined and transferred to percentages. Microscopy was performed under immersion at 90×10 magnification.

2.3 Pathomorphological studies

For the pathomorphological studies the skin flaps were taken from the lateral parts of the body. The conventional methods of the pathomorphological analysis were used. The material was fixed in 10% neutral formalin, conducted through a battery of alcohols with an ascending concentration (70, 80, 96I, 96II, 96II, 100°), alcohol-chloroform, chloroform, chloroform-paraffin (at 37°C), paraffin (at 57°C) and poured in paraffin wax. The serial paraffin sections (7μm thickness) were stained with hematoxylin and eosin, and van Gieson staining. The content of fibroblasts, macrophages and neutrophils (arbitrary units) was calculated on an area of 130×130μm of the histological section where each cell type was counted in 20 fields of the vision followed by dividing the number of each cell type by the number of vision fields.

2.4 Western blot analysis

The levels of nuclear factor κB (NF-κB) p65 subunit, phosphorylated at Ser 311, vascular endothelial growth factor (VEGF), and the VEGF receptor 1 (Flt-1) in regenerated connective tissue were determined by western blot analysis. Total protein extracts were prepared from frozen skin samples (200–250 mg) using standard protocol with RIPA buffer (20 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1% Triton X-100; 1 mM EGTA; 0.1% SDS, 1% sodium deoxycholate, 10 mM sodium pyrophosphate). Briefly, skin samples (100 mg) were lysed for 20 min in RIPA buffer in the presence of protease inhibitor cocktails (PIC, Sigma, USA), then sonicated and centrifuged for 20 min (14000 g) at 4°C. The protein extracts were mixed with Laemlly buffer (150 mM Tris-HCl (pH 6.8), 1% SDS, 0.3% bromphenol blue, 20% glycerol) and heated at 95°C for 5 minutes. The concentration of total protein was measured spectrophotometrically using the Stoscheck method with modifications on the spectrophotometer SF-2000.

Total protein fractions (70μg) were subjected to SDS-PAGE and blotted on nitrocellulose membranes. Immunoblotting was conducted with primary antibodies against VEGF (Merck, Germany), NF-κB p65, phosphorylated at Ser 311 (Santa Cruz, USA), IκB-α (Santa Cruz, USA) Ta Flt-1 (Santa Cruz, USA), β-actin (Sigma, USA). Anti-IκB-α and anti-Flt-1 antibodies were diluted 1:500, anti-VEGF antibody was diluted 1:2000, anti-pNF-κB antibody was diluted 1:200 in PBS supplemented with 0.1% (vol./vol.) Tween and 5% (wt./vol.) non-fat milk. Primary-antibody-bound membranes were incubated with peroxidase-conjugated secondary antibodies: anti-mouse IgG (Fab Specific)-Peroxidase (Sigma, USA) 1:5000 or anti-rabbit IgG (H + L)-HRP conjugate (Bio-Rad Laboratories, Inc., USA) 1:2500. Thereafter, the membranes were developed with chemiluminescent agents: p-coumaric acid (Sigma, USA) and luminol (AppliChem GmbH, Germany). Tissue levels of all proteins were normalized to β-actin. The immunoreactive bands were quantified with Gel-Pro Analyzer v3.1 and TL-120 (TotalLab Ltd., USA).

2.5 Statistical analysis

Statistical data processing was performed using the statistical package IBM SPSS Statistics 23. Calculated the Pearson correlation coefficient (R), with a p-value, 95% confidence interval for the correlation coefficient. The Shapiro-Wilk test was used to check the normality distribution. Most of the obtained data had a distribution close to Gaussian, so to compare the indicators at different periods of observation, a multiple comparison criterion was used - the Scheffer criterion. For nonparametric data, we used the criterion χ² and Kruskal-Wallis. The charts were provided in the form of columns indicating (CI 95%) or Box-and-Whisker plot. It was believed that the data differ by P < 0,05.

3 Results and discussion

After thermal injury by copper plates, burn wounds in the form of an ellipse are symmetrical on appearing on both exposed parts of the rat body. On day 3, 7, 14, and 21 day bone marrow, newly formed connective tissue and the blood cell composition was analysed in control rats and rats suffering from diabetes (Fig. 1).

Fig. 1

The design of this study. Mature granulocytes and monocytes were analysed from bone marrow (Gr_BM: mature granulocytes). In the newly generated tissue the numbers of Mon (monocytes), Neutr (neutrophils), Macroph (macrophages), Fibrobl (fibroblasts), and the expression of pNF_kB (nuclear factor κB), VEGF (vascular endothelial growth factor), Flt-1 (receptor of VEGF) was analyzed. From peripheral blood, neutophils (Neu_PB) and monocytes (Mon_PB) were quantified.

Visual assessment of wounds did not reveal any significant difference between the animals without somatic pathology and in case of the composition applications (Fig. 2). Burn injury causes inflammation, which is manifested by hyperemia, swelling and cell infiltration, followed by scab formation on the wound surface. Signs of coagulation necrosis were detected on day 3 of observation. By day 14, the wound was devoid of epithelial lining and covered with a scab and areas of fibrinoid tissue necrosis. On day 21, the epithelialization of the damaged surface was observed, but the central zone remained without epithelial lining and was covered with a scab (Fig. 2) [19].

Fig. 2

Burn wound appearance of the control (top) and hyperglycemic (bottom) rats in the dynamics of healing (days 3, 7, 14, 21).

In the regenerated connective tissue, we investigated the level of phosphorylated p65 subunit on Ser311 of the nuclear factor kappa B (NF-κB) and vascular endothelial growth factor VEGF, VEGFR-1 receptor (Flt-1) the expression in control rats group without somatic pathology and under conditions of hyperglycemia in the dynamics of burn wound healing (on the 3, 7, 14, 21 day) using western blots (Fig. 3A). Most obviously, the VEGF protein expression seems to be reduced in the hyperglycemic rats (H) compared to the controls, particularly at day 3 and day 21 (Fig. 3A). The quantification of the western blot data revealed that pNF-κB and VEGF are decreased in the hyperglycemic rats over the whole observation period (Fig. 3B). The FLT-1 expression was higher at day 3 and day 14 in hyperglycemic rats, whereas the opposite was observed at day 7 and day 21 (Fig. 3B).

Fig. 3

Representative immunoblots are shown (A) and quantified using β-actin as a loading control for tissue lysates. The bar graphs of protein content (B) are presented as means±SEM (n = 6/group) of pNF-κB (left), VEGF (middle), FLT1 (right) in the regenerated connective tissue of the rat skin in the dynamics of burn wound healing (3, 7, 14, and 21 day) in the control group without somatic pathology (light bars), under conditions of stable uncorrected hyperglycemia (dark bars). ^*Indicates significant differences compared with the control (P < 0.001). The charts were provided in the form of columns indicating (CI 95%). All experiments were duplicated.

The results of our studies showed (Fig. 4) that animals of all groups in the first 3 days after burn had necrotic changes in the epidermis, hair follicles, dermis edema. The stasis and thrombosis were developed in the vessels of hemomicrocyrculatory channel. In the control group of the animals, the burn wound infliction has led to the development of a young granulation tissue rich in neutrophilic granulocytes at the bottom of the wound during the first 3 days of the observation. In 3 days of the experiment, a large number of the cellular elements has accumulated in the edges and bottom of the wound that form a full granulation tissue by the 7th day. On the 14th day of observation, there was a significant amount of collagen fibers the density of which has increased from the surface to the deep layers. The regeneration of the epidermis occurred simultaneously with the connective tissue regeneration.

Fig. 4

Content of neutrophilic granulocytes (left), macrophages (middle), and fibroblasts (right) (arbitrary units) are presented as means±SEM (n = 6/group) in the connective tissue regenerate of the derma during the dynamics of the burn wound skin in the rats without somatic pathology – control group (light bars) under conditions of stable uncorrected hyperglycemia (dark bars). ^*Indicates significant differences compared with the control (P < 0.001). The charts were provided in the form of columns indicating (CI 95%). All experiments were duplicated. The content of fibroblasts, macrophages and neutrophils (arbitrary units) was calculated on an area of 130 × 130 μm of the histological section where each cell type was counted in 20 fields of the vision followed by dividing the number of each cell type by the number of vision fields.

Thus, during the entire period of wound healing, the content of the neutrophils has gradually decreased in the newly formed connective tissue but the content of the macrophages and fibroblasts has increased by the 7th day decreasing in the following days of observation in the control group of animals.

Under conditions of physiological wound healing (Table 1, 2, and 3), we found a correlation between blood cells of neutrophils and monocytes (R = 0.805, p < 0.01), as well as their followers in the tissue of neutrophils and macrophages (R = 0.879, p < 0.01).

Table 1

Correlation (Pearson’s ratio) matrix highlighting the wound healing dynamics of bone marrow cells, peripheral blood, and regulatory molecules in control and hyperglycemic rats

Fabric parameters		Bone marrow				Peripheral blood
		Gr_BM		Mon_BM		Neu_PB		Mon_PB
		Control	Hyperglyc	Control	Hyperglyc	Control	Hyperglyc	Control	Hyperglyc
BM	Gr_BM	1	1	0.297	–0.755^**	–0.269	–0.752^**	–0.005	–0.071
BM	Mon_BM	0.297	–0.755^**	1	1	–0.335	0.649^**	–0.159	0.112
PB	Neu_PB	–0.269	–0.752^**	–0.335	0.649 ^**	1	1	0.805^**	0.026
PB	Mon_PB	–0.005	–0.071	–0.159	0.112	0.805 ^**	0.026	1	1
CT	Neutr_Tissue	–0.065	–0.688^**	0.049	0.596 ^**	0.131	0.717 ^**	0.507 ^*	0.012
CT	Macrophages	–0.356	–0.859^**	0.003	0.616 ^**	0.041	0.622 ^**	0.273	0.183
CT	Fibroblast	–0.748^**	–0.181	–0.335	0.096	0.210	–0.071	–0.152	0.168
CT	pNF_kB	0.687 ^**	0.738 ^**	0.134	–0.533^**	–0.205	–0.506^*	–0.119	–0.032
CT	VEGF	–0.342	0.038	–0.330	–0.110	0.708 ^**	–0.364	0.304	0.126
CT	FLT1	0.259	0.118	–0.086	–0.092	0.472 ^*	–0.349	0.242	0.169

BM: Bone Marrow, PB: Peripheral Blood, CT: Connective Tissue, Gr_BM: mature granulocytes, Mon: monocytes, Neutr: neutrophils, Macroph: macrophages, Fibrobl: fibroblasts, pNF_kB: nuclear factor κB, VEGF: vascular endothelial growth factor, Flt-1: receptor of VEGF. ^*: p < 0.05; ^**: p < 0.01.

Table 2

Correlation (Pearson’s ratio) matrix of neutrophils, macrophages and fibroblast comparing control and hyperglycemic rats

Fabric parameters		Neutr_Tissue		Macrophages		Fibroblast
		Control	Hyperglyc	Control	Hyperglyc	Control	Hyperglyc
BM	Gr_BM	–0.065	–0.688^**	–0.356	–0.859^**	–0.748^**	–0.181
BM	Mon_BM	0.049	0. 596^**	0.003	0. 616^**	–0.335	0.096
PB	Neu_PB	0.131	0.717^**	0.041	0. 622^**	0.21	–0.071
PB	Mon_PB	0. 507^*	0.012	0.273	0.183	–0.152	0.168
CT	Neutr_Tissue	1	1	0. 879^**	0. 798^**	–0.203	–0.436^*
CT	Macrophages	0. 879^**	0. 798^**	1	1	0.172	–0.050
CT	Fibroblast	–0.203	–0.436^*	0.172	–0.050	1	1
CT	pNF_kB	–0.519^**	–0.209	–0.780^**	–0.558^**	–0.633^**	–0.545^**
CT	VEGF	–0.454^*	–0.616^**	–0.351	–0.238	0.562^**	0. 800^**
CT	FLT1	–0.665^**	0.294	–0.818^**	0.262	–0.099	–0.506^*

Table 3

Correlation (Pearson’s ratio) matrix of pNF-kB, VEGF, and FLT1 comparing control and hyperglycemic rats

Fabric parameters		pNF-kB		VEGF		FLT1
		Control	Hyperglyc	Control	Hyperglyc	Control	Hyperglyc
BM	Gr_BM	0.687^**	0.738^**	–0.342	0.0380	0.259	0.118
BM	Mon_BM	0.134	–0.533^**	–0.33	–0.110	–0.086	–0.092
PB	Neu_PB	–0.205	–0.506^*	0.708^**	–0.364	0. 472^*	–0.349
PB	Mon_PB	–0.119	–0.032	0.304	0.126	0.242	0.169
CT	Neutr_Tissue	–0.519^**	–0.209	–0.454^*	–0.616^**	–0.665^**	0.294
CT	Macrophages	–0.780^**	–0.558^**	–0.351	–0.238	–0.818^**	0.262
CT	Fibroblast	–0.633^**	–0.545^**	0. 562^**	0. 800^**	–0.099	–0.506^*
CT	pNF_kB	1	1	–0.118	–0.443^*	0. 606^**	0. 445^*
CT	VEGF	–0.118	–0.443^*	1	1	0. 665^**	–0.377
CT	FLT1	0.606^**	0. 445^*	0.665^**	–0.377	1	1

We already described an association (R = 0.687, p < 0.01) of pNF-κB production in the regenerated connective tissue of the skin in the control group with the relative content of bone marrow (BM) neutrophils - mature granulocytes, between the relative content of neutrophils and the level of VEGF, R > 0 (R = 0,708, p < 0,01), and between the relative content of PB neutrophils and the expression level of the Flt-1 receptor (R = 0,472, p < 0,05) (Fig. 4) [18].

We claimed that NF-κB induces the production of growth factors for myeloid progenitor cell. Thus, humoral signals entering the bone marrow stimulate the differentiation of neutrophilic granulocytes and their release into the blood. Neutrophils, which enter the damaged area of the burn wound, express endothelial growth factors such as VEGF and regulate the expression of receptors for these ligands in the connective tissue regenerate, thereby contributing to the restoration of regulated angiogenesis as an important factor in timely physiological repair [20].

In this work, we deepened the understanding of the influence of tissue infiltrating cells on the links of angiogenesis. We found that there is a correlation between VEGF content and fibroblasts (R = 0.562, p < 0.01), but there is no correlation between fibroblasts and the VEGF receptor in tissue. We also found a negative association between the content of VEGF and the number of neutrophils in the regenerate (R = –0.454, p < 0.01), just like between the neutrophils infiltrating the regenerated tissue and the expression of the FLT-1 receptor (R = –0.665, p < 0.01). This confirms our hypothesis that blood neutrophils are the main donors of VEGF and stimulate the expression of the FLT-1 receptor, which is the basis for the neoangiogenesis and subsequently optimal wound healing.

In the healing process of control rats, macrophages are also not associated angiogenesis induction. On the contrary, they suppress neoangiogenesis as the main “wipers” of tissue. We found a negative association of macrophages with VEGF content (R = –0.351, p > 0.05), and a negative one with FLT-1 receptor content (R = –0.818, p < 0.01). Under conditions of hyperglycemia, the picture of the correlation changes substantially.

Marks as in Table 1, 2 and 3 draw attention to the fact of negative association between granulocytes of BM and tissue populations of cells: neutrophils (R = –0,688, p < 0,01) and macrophages (R = –0,859, p < 0,01). At the same time, there is a high positive correlation between BM granulocytes and NF-κB transcription factor. This emphasizes our hypothesis that under hyperglycemia, activation of intranuclear transcription processes that stimulate proliferation in the bone marrow, occurs. However, granulocytes do not mature and their functional capacity is low for ensuring regeneration (Fig. 6).

Fig. 5

Comparison of indicators in control rats are presented as means±SEM (n = 6/group): A: mature granulocytes bone marrow (columns, left scale) and pNF-κB content of connective tissue (line, right scale); B: peripheral blood neutrophils (columns, left scale) and VEGF content of connective tissue (line, left scale). The charts were provided in the form of columns indicating (CI 95%). All experiments were duplicated.

Fig. 6

Comparison of the indicators in hyperglycemic rats are presented as means±SEM (n = 6/group): A: peripheral blood neutrophils (columns, left scale) and VEGF content of connective tissue (line, right scale); B: neutrophils of connective tissue (columns, left scale) and VEGF content of connective tissue (line, left scale). The charts were provided in the form of columns indicating (CI 95%). All experiments were duplicated.

At the same time, on in hyperglycemic rats, we found an association of BM monocytes with blood neutrophils (R = 0.649, p < 0.01), tissue infiltrating neutrophils (R = 0.596, p < 0.01), with macrophages (R = 0.616, p < 0.01). There was an association between blood neutrophils and cells in the regenerated tissue: neutrophils (R = 0.717, p < 0.01) and macrophages (R = 0.622, p < 0.01), as well as the association between neutrophils and macrophages 0.798, p < 0.01). We consider, that this is expected and could be a sign of chronic inflammation and increased transmigration of blood cells into regenerating burn wound throughout the observation period.

In hyperglycemic rats, we found a correlation between fibroblasts in the regenerated tissue and VEGF (R = 0.800, p < 0.01), whereas a negative association between fibroblasts and FLT-1 (R = –0.506, p < 0,05) was found.

Thus, we can assume, that under conditions of physiological regeneration, the main trigger for the stimulation of angiogenesis in the tissue is the migration of neutrophils from the peripheral blood to the damaged tissue. At the same time, inflammation in the burned tissue and the destruction of cellular structures contribute to the fact that cells surrounding the area of inflammation show increased NF-κB activity. Gene transcription leads to the synthesis of cytokines, active molecules, and growth factors including G-CSF [21]. This stimulates the proliferation in the bone marrow of granulocytes. Their release from the bone marrow into the bloodstream is followed by enhanced migration into the burned area close the signal chain and provide controlled angiogenesis [22].

Under hyperglycemic conditions, the sequence of events appears to be different. Transcription processes in cells of the regenerated tissue stimulate the bone marrow due to growth factor. However, in the bone marrow cell differentiation is delayed and they lose their functional capacity. The release of mature granulocytes into the blood and their migration into the damaged tissue is impaired. Deficiency of mature neutrophils reduces the content of VEGF in the burned tissue. Along with this in the regenerated tissue there is an imbalance of the receptor and ligand associated with angiogenesis: VEGF and FLT-1, which also contribute to the disruption of the new tissue creation, enhance hypoxia and inhibit the restoration of tissue architecture. This prevents the physiological course of burn wound healing, leads to pathological changes in blood vessels, delays and disrupts the restoration of connective tissue architecture, and can be an important factor in the deterioration of tissue regeneration in diabetes.

Diabetes affects both injury and repair processes in a manner distinct from other vascular diseases. VEGF expression changes paradoxically with diabetes, it increases in the retina and renal glomeruli, but it decreases in the myocardium, peripheral limbs, and nerves correlating with the extent of angiogenesis. The study of connective tissue remodeling processes that provide quality wound healing on the background of hyperglycemia can be a theoretical basis for the creation of rational pharmacological treatment and prevention regimens in patients with local thermal damage and diabetes.

In chronic hyperglycemia, tissue extracellular matrix proteins undergo modification due to the formation of advanced glycation end products (AGEs). Matrix AGEs increase oxidative stress through their signaling function. It includes interaction with RAGE receptors, activation of NADP-oxidase and xanthine oxidase, which are powerful sources of reactive oxygen species (ROS). They contribute to pro-inflammatory and pro-thrombotic changes in the wound healing process, prolongation of its inflammatory stage.

For example, we have studied the application of exogenous proteases to be theoretically substantiated pharmacological approach to promoting the cleavage of disintegration products in necrotic wound tissue (debridement), providing a substantial change in the microenvironment of residents connective tissue macrophages, fibroblasts, and epithelial cells. We found out, use of exogenous proteases is appropriate to enhance proteolysis in tissues with predominance of glycated proteins in case of chronic hyperglycemia to ensure controlled proteolysis [19].

Barrett et al. [23] described both the general molecular processes involved in diabetic microvascular disease and many of their tissue-specific expressions. They showed, that our ability to successfully intervene to prevent or reverse microvascular disease is quite limited [23]. Insights gained by the use of newer tools, including genetic, proteomics, metabolomics, and other analyses, will certainly add new insights in the basic functioning of microvascular cells, and these insights will light the way to improved therapy.

4 Conclusions

Under conditions of hyperglycemia cell differentiation in the bone marrow is delayed, the release of mature granulocytes into the blood, and their migration into the tissue is reduced compared to non-diabetic control rats. The regeneration of the burned tissue shows an imbalance of the receptor and ligand of angiogenesis: VEGF and FLT-1, which can deepen hypoxia, prevent the physiological course of burn wound healing, delay, and disrupt the restoration of connective tissue architecture.

Conflicts of interest

The authors declare that they have no conflicts of interest.

Funding

This work was supported by a research grant of the Ministry of Health of Ukraine (No state registration 0119U101219) and the German Federal Ministry of Education and Research by the grant TriDiMed (01DK20008).

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