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Synthesis of hyaluronic acid. The structure and use of hyaluronic acid in medicine. Physiological role of hyaluronic polymers

Hyaluronic acid [HA] is found in the extracellular matrix of vertebrate tissues, in the surface coatings of certain Streptococcus species and the pathogenic bacterial Pasteurella microorganisms, and on the surface of some partially viral algae. Hyaluronic acid synthases [HA] are enzymes that polymerize HA using UDP-sugar precursors that are found in the outer membranes of these organisms. GKS genes were identified from all of the above sources. There seem to be two distinct classes of GCS based on differences in amino acid sequence, predicted topology in the membrane, and putative reaction mechanism.

All GCS were identified as class I synthases, with the exception of GCS in the species Pasteurella. The catalytic mode of operation of a single class II GCS (PMCS) was also explained. This enzyme lengthens external HA-linked oligosaccharide acceptors by adding individual monosaccharide units to the non-decreasing end to form long polymers in vitro; no class I GCS has this ability. The method and direction of HA polymerization catalyzed by GCS class I remain unclear. The enzyme pmGCS was also analyzed for its two activities: GlcUA-transferase and GlcNAc-transferase. Thus, two active sites exist in one pmGCS polypeptide, refuting the widely accepted dogma of glycobiologists: "one enzyme - one modified sugar". Preliminary evidence suggests that Class I enzymes may also have two sites of activity.

The catalytic potential of the enzyme pmGCS can be used to create new polysaccharides or to design oligosaccharides. Because of the myriad potential HA-based medical therapies, this chemoenzymatic technology promises to be beneficial in our pursuit of good health.

Keywords

Hyaluronic acid (HA), chondroitin, glycosyltransferase, synthase, catalysis, mechanism, chimeric polysaccharides, monodisperse oligosaccharides

Introduction

Hyaluronan [HA] is a very rich glucosaminoglycan in vertebrates with both structural and signaling roles. Certain pathogenic bacteria, namely groups A and C of Streptococcus and type A of Pasteurella multocida, produce an extracellular covering HA called a capsule. In both types of HA, the capsule is a toxicity factor that provides bacteria with resistance to phagocytes and complementarity. Another HA-producing organism is the seaweed chlorella infected with a specific large double-stranded DNA virus, PBCV-1. The role of HA in the life cycle of this virus is not yet clear at this time.

Figure 1. Reaction of HA biosynthesis.

Enzymes of the class of glycosyltransferases that polymerize HA are called HA synthases (or GCS), according to the old terminology, which also includes HA synthases. All known HA synthases are species of a single polypeptide responsible for the polymerization of the HA chain. UDP sugar precursors, UDP-GlcNAc, and UDP-GlcUA are used by HA synthases in the presence of a divalent cation (Mn and / or Mg) at neutral pH (Fig. 1). All synthases are membrane-bound proteins in a living cell and are found in the membrane fraction after cell lysis.

Between 1993 - 1998 were identified and cloned at the molecular level HA synthases of groups A and C Streptococcus [cGCS and seHCS, respectively], HA synthases of vertebrates [GCS 1,2,3], HA synthase of algal virus [cGCS], and also HA-synthase type A of the species Pasteurella multocida [pmGCS]. The first three types of HA synthases appear to be very similar in size, amino acid sequence, and predicted topology in the membrane. On the other hand, the HA synthase of the species Pasteurella is larger and has a sequence and predicted topology that is significantly different from other synthases. Therefore, we assumed the existence of two classes of HA synthases (Table 1). Class I enzymes include streptococcal, vertebrate and viral proteins, while Pasteurella is currently the only member of Class II. We also have some evidence that the catalytic processes of Class I and Class II enzymes are different.

Table 1. Two classes of HA synthases:

Although Pasteurella HA synthase was the last enzyme to be discovered, certain features of PMCS contributed to significant advancements in its study compared to some members of Class I enzymes, which have been studied for four decades. A key feature of PMGC, which made it possible to clarify the molecular direction of polymerization and the identification of its two active sites, is the ability of PMGC to lengthen the externally located acceptor oligosaccharide. Recombinant pmGCS adds single monosaccharides in a repeated manner to the HA-associated oligosaccharide in vitro. The intrinsic feature of each monosaccharide transfer is responsible for forming an alternative repeat of disaccharides in this glucosaminoglycan; simultaneous formation of the disaccharide unit is not required. On the other hand, no such elongation of external acceptors has been proven for any class I enzyme. Through basic scientific research, we have now developed some biotechnological applications of the remarkable protein of the Pasteurella HA synthase class.

Materials & methods

Reagents

All reagents for molecular biology research, without special labeling, were from Promega. Standard oligonucleotides were from the Great American Gene Company. All other high purity reagents, unless otherwise noted, were from Sigma or Fisher.

Truncation of PMGCS and point mutants

A number of truncated polypeptides were produced by amplification of the pPm7A insert by polymerase chain reaction with Taq polymerase (Fisher) and synthetic oligonucleotide primers corresponding to different parts of pmGCS, with an open reading frame. The amplicons were then cloned into the expression plasmid pKK223-3 (tac promoter, Pharmacia). The resulting recombinant constructs were transformed into Escherichia coli strain TOP 10F "(Invitrogen) and grown in LB medium (Luria-Bertani) with ampicillin selection. Mutations were made using the QuickChange method of site-directed mutagenesis (Stratagene) with the pKK / pmGKS plasmid as sample.

Enzyme preparation

To prepare a membrane containing full length recombinant pmGCS, pmGC1-972 was isolated from E. coli as described. For the soluble truncated pmGCS proteins, pmGCS1-703, pmGCS1-650, and pmGCS1-703 — containing mutants, cells were recovered using B-PerTM II Bacterial Protein Extraction Reagent (Pieree) according to the manufacturer's instructions, except that the procedure was performed with 7 ° C in the presence of protease inhibitors.

Enzymatic pathways for HA polymerization. GlcNAc modification or GlcUA modification

Three options were developed to detect whether (a) the polymerization of long HA chains occurs or (b) the addition of a single GlcNAc to the GlcUA-terminal HA acceptor oligosaccharide, or (c) the addition of a single GlcUA to the GlcNAc-terminal HA acceptor oligosaccharide. The total GCS activity was estimated for a solution containing 50 mM Tris, pH 7.2, 20 mM MnCl2, 0.1 M (NH4) 2SO4, 1 M ethylene glycol, 0.12 mM UDP- (14C) GlcUA (0.01 μCi; NEN), 0.3 mM UDP- GlcNAc and a different set of HA oligosaccharides obtained from testicles by treatment with hyaluronidase [(GlcNAc-GlcUA) n, n = 4-10] at 30 ° C for 25 minutes in a reaction volume of 50 μl. GlcNAc transferase activity was assessed for 4 minutes in the same buffer system with a different set of HA oligosaccharides, but with only one sugar as a precursor - 0.3 mM UDP- (3H) GlcUA (0.2 μCi; NEN). GlcUA transferase activity was assessed for 4 minutes in the same buffer system, but only with 0.12 mM UDP- (14C) GlcUA (0.02 μCi) and with an odd set of HA oligosaccharides (3.5 μg uronic acid) prepared by exposure to acetate mercury to HA-lyase Streptomyces. Reactions were stopped by adding SDS up to 2% (w / v). The reaction products were separated from the substrates by paper chromatography (Whatman 3M) ​​with ethanol / 1M ammonium sulfate, pH 5-5 as main solvent (65:35 for GCS and GlcUA-Tase; 75:25 for GlcNAc-Tase). To evaluate the GCS, a sample of the paper strip was washed with water, and the pooling of radioactive sugars in the HA polymer was detected by liquid scintillation calculated using the BioSafe II cocktail (RPI). For half-assay reactions, the sample and 6 cm downstream bands were counted in 2 cm chunks. All evaluation experiments were calculated to be linear with incubation time and protein concentration.

Gel filtration chromatography

The HA size of the polymers was analyzed by chromatography on Phenomenex PolySep-GFC-P 3000 columns, elution was performed with 0.2 M sodium nitrate. The column was standardized with different sizes of fluorescent dextrans. The radioactive components were detected using the LB508 Radioflow sensor (EG & G Berthold) and the Zinsser cocktail. Compared to a full GCS assessment using paper chromatography described above, these 3 minute reactions contained twice UDP sugar concentrations, 0.06 μCi UDP- (14C) GlcUA, and 0.25 nanograms of a range of HA oligosaccharides. In addition, the addition of boiling (2 minutes) ethylenediamine tetracylic acid (22 mM final concentration) was used to terminate the reactions instead of adding SDS.

Results and discussion

Utilization and specificity of the GCS acceptor

Several oligosaccharides have been tested as acceptors for recombinant pmGKS1-972 (Table 2). HA oligosaccharides were obtained from testicles by hyaluronidase cleavage and lengthened with pmGCS using delivered suitable UDP sugars. Reduction with sodium borohydrate does not interfere with the activity of the acceptor. On the other hand, oligosaccharides obtained from HA by cleavage with lyase do not support elongation; dehydrated unsaturated unreduced terminal GlcUA residues require hydroxyl groups that can attach the incoming sugar from the UDP precursor. Therefore, pmGCS-catalyzed elongation occurs in the case of unreduced end groups. In a number of parallel experiments, recombinant forms of class I synthases, cGCS and x1HCS, were found that do not lengthen the HA-derived acceptors. Considering the direction of activity of class I enzymes, conflicting reports have been made and further research is needed.

Table 2. Specificity of oligosaccharide acceptoro pmGCS:

Interestingly, chondroitin sulfate pentamer is a good acceptor for PMGS. Other structurally related oligosaccharides, such as chitotetrose or heparosan pentamer, do not, however, serve as acceptors for PMGCS. In general, pmGCS seems to require β-linked GlcUA-containing acceptor oligosaccharides. We hypothesize that the oligosaccharide binding site is intermediate in the HA retention chain during polymerization.

Molecular analysis of pmGCS transferase activity: two active sites in one polypeptide

The ability to measure two components of the glycosyltransferase activity of GK synthase, GlcNAc-transferase and GlcUA-transferase, allowed the molecular analysis of pmGCS. We noted that a short duplicated sequence motif: Asp-Gly-Ser (Aspartic acid-ta-Glycine-Serine) was present in PMGCS. From an analysis comparing the hydrophobic groups of many other glycosyltransferases that produce β-linked polysaccharides or oligosaccharides, it has been suggested that there are generally two types of domains: regions "A" and "B". PMGKS, a class II synthase, is unique in that it contains two "A" domains (personal communication, B. Henrissat). It has been proposed that certain members of class I HA synthases (cGKS) contain single "A" and single "B" regions. Various deletions or point mutants of pmGCS were evaluated for their ability to polymerize HA chains or their ability to add a single sugar to the HA acceptor oligosaccharide (Table 3). To summarize, PMCS contains two distinct active sites. Mutagenesis of the DGS motif aspartate (residue 196 or 477) at both sites resulted in a loss of HA polymerization, but the activity of the other site remained relatively unaffected. Thus, the dual activity of HA synthase was converted into two distinct single actions of glycosyltransferase.

Table 3. Activity of PMGCS with a deleted site or point mutation.

Removal of the last 269 residues from the terminal carboxyl group converted a weakly expressed membrane protein into a well-expressed soluble protein. Examination of the amino acid sequence of the PMCS protein in this area, however, does not show the typical features of the secondary structure that would provide a direct interaction of the enzyme with the lipid bilayer. We hypothesize that the terminal carboxyl group of the catalytic enzyme pmGC is docked with the membrane-bound membrane-bound polysaccharide transport apparatus of the living bacterial cell.

The first "A" region of pmGCS, A1, is the GlcNAc pelvis, while the second "A" region, A2, is the GlcUA pelvis (Fig. 2). This is the first identification of two active sites for an enzyme that produces a heteropolysaccharide, as well as clear evidence that one enzyme can actually transfer two different sugars. An enzyme of the species P. multocida, different from type F, called PMCS, was found and it was found that it catalyzes the formation of the nonsulfatable polymer chondroitin. HA and chondroitin are identical in structure, except for the aforementioned polymer, which contains N-acetylglucosamine instead of GlcNAc. Both PMCS and PMCS are 87% identical at the amino acid level. Most of the changes in the residues are in the A1 region, which is quite consistent with the hypothesis that this region is responsible for the transfer of hexosamine.

Figure 2. Schematic representation of PMGC areas.
Two independent transferase domains, A1 and A2, are responsible for catalysing the polymerization of the HA chain. Repetitive, sequential additions of single sugars quickly build the HA chain. It seems that the carboxyl end of pmGCS interacts in some way with the membrane-bound transport apparatus of the bacterial cell.

Figure 3. Model of HA biosynthesis using PMGCS.
Single sugars are added to each "A" domain in a repetitive fashion to the non-reducing end of the HA chain. The intrinsic precision of each step of transferase activity maintains the repeat structure of the HA disaccharides. The resulting HA chain is probably retained by pmGCS during catalysis through the oligosaccharide-binding site.

We have demonstrated efficient transfer of a single sugar by PMGCS in vitro by several types of experiments, therefore, we hypothesized that HA chains are formed by the rapid, repeated addition of a single sugar by class II synthase (Fig. 3). To date, one line of evidence suggests that the class I enzyme also possesses two transferase sites. A mutation of leucine residue 314 to valine in mmGCS1, in a part of the GlcUA-pelase pre-site, has been reported to convert this vertebrate CGS into chito-oligosaccharide synthase. No site with corresponding GlcNAc transferase activity has been identified.

Polymer grafting with polysaccharide synthases: adding HA to molecules or solid particles

Researching PMGCS in a research lab has transformed the concept of HA synthases from the kingdom of difficult, stubborn animal-like monsters to potential biotech workhorses. New molecules can be formed using the ability of PMGC to graft long HA chains onto short HA-derived chains or chondroitin-derived acceptors. For example, useful acceptors can consist of small molecules or drugs with covalently linked HA or chondroitin oligosaccharide chains (4 sugars long, for example). Alternatively, HA chains can be added to an oligosaccharide primer immobilized on a solid surface (Table 4). Thus, long HA chains can be gently added to sensitive substances or delicate devices.

In another application, new chimeric polysaccharides can be formed because the use of PMGC by an oligosaccharide acceptor is not as stringent as the saccharide transferase specificity. Chondroitin and chondroitin sulfate are recognized as acceptors of PMGCS and are extended by HA chains of various lengths (Fig. 4). On the contrary, PMCS is very homologous to chondroitin synthase; it recognizes and lengthens HA acceptors by chondroitin chains. Chimeric glucosaminoglycan molecules are formed containing natural, distinct bond compounds. These grafted polysaccharides can serve to attach to a cell or tissue that binds HA to another cell or tissue that binds chondroitin or chondroitin sulfate. In certain aspects, the grafted glucosaminoglycans resemble proteoglycans, which are essential matrix components in vertebrate tissues. But since no protein linkers are present in the chimeric polymers, the antigenicity and proteolysis problems surrounding the medical use of proteoglycans are eliminated. The risk of transmission of infectious agents by tissues extracted from animals to a human patient is also reduced by the use of chimeric polymers.

Table 4. PMGCS-initiated grafting of HA onto polyacrylamide beads. The reaction mixture contains radiolabeled PMGCS UDP- (14C) GlcUA and UDP- (3H) GlcNAc, as well as various immobilized sugar primers (acceptors coupled by reductive amination to aminobusins) were presented. The beads were washed and radioactively incorporated onto other beads as measured by liquid scintillation calculation. HA chains were grafted onto plastic beads using a suitable primer and PMGCS.

Figure 4. Schematic representation of grafted polysaccharide structures. HA synthase of the species Pasteurella or chondroitin synthase will elongate certain other polymers at the non-reducing end in vitro to form new chimeric glucosaminoglycans. Some examples are shown.

Synthesis of monodisperse HA and HA-linked oligosaccharides

In addition to adding a large polymeric HA chain to the acceptor molecules, PMGCs synthesize certain smaller HA oligosaccharides ranging from 5 to 24 sugars. Using the wild-type enzyme and various reaction conditions, a HA oligosaccharide containing 4 or 5 monoaccharides, extended with several sugars to longer versions, which are very often difficult to obtain in large quantities, has been relatively easily obtained. We found that combining the soluble GlcUA-Tase mutant and the soluble GlcNAc-Tase mutant in the same reaction mixture allows the formation of the HA polymer if the system is equipped with an acceptor. Within 3 minutes, a chain of about 150 sugars (-30 kDa) was made. Any single mutant synthase will not form a HA chain as a result. Therefore, if further control of the reaction is done by selectively combining various enzymes, UDP sugars and acceptors, then certain monodisperse oligosaccharides can be obtained (Fig. 5).

Figure 5. Preparation of certain oligosaccharides.
In this example, the HA acceptor tetrasaccharide is extended by a single chondroitin disaccharide unit using two steps with an immobilized Pasteurella synthase mutant (shown by white arrows). The product shown is the new hexasaccharide. Repetition of the cycle again produces the oligosaccharide, two cycles form the decaccharide, etc. If the acceptor was previously linked to another molecule (eg drug or drug), then the new conjugate would be extended with a short HA, chondroitin, or a hybrid chain as desired.

For example, in one embodiment, a mixture of UDP-GlcNAc, UDP-GlcUA and an acceptor is continuously circulated through separate bioreactors with immobilized mutant synthases that transfer only a single sugar. With each incubation cycle of the bioreactor, a different sugar group is added to the acceptor to form small, specific HA oligosaccharides. The use of a similar PMCS mutant (for example, GalNAc-Tase) in one of the steps allowed the formation of mixed oligosaccharides using UDP-GlcNAc. The biological activity and therapeutic potential of small HA oligosaccharides is a complex area of ​​research that will require certain, monodisperse sugars for unambiguous interpretation.

Conclusion

Obviously, there are two different classes of HA synthases. The best characterized is the class II enzyme of the Pasteurella species, which lengthens the HA chain by repeated addition of a single sugar to the non-reducing end of the HA chain. The direction and mode of operation of class I synthases (streptococcal, viral, and vertebrate enzymes) remain unclear. In applied sciences, the ability of PMGC to lengthen exogenously located acceptor molecules is useful for creating new molecules and / or devices with potential medical applications.

Structure

Molecule hyaluronic acid looks like a long ribbon built of alternating sugars - D-glucuronic acid and N-acetylglucosamine. forming the basic disaccharide unit ( rice. one).

Fig. 1. Hyaluronic acid is composed of alternating disaccharide units

One chain can contain up to 250 thousand disaccharide units. The molecular weight of this natural polysaccharide reaches 10 thousand kDa. HA is part of the synovial fluid, vitreous body, found in the umbilical cord, cornea, bones, heart valves, oocyte membranes.

Of fundamental importance is the property hyaluronic acid(HA) bind and retain (due to hydrogen bonds) a large amount of water: 1 HA molecule binds 200-500 water molecules. At the same time, it has the effect of a "diaper" - it does not give up water even when its content in the environment decreases. The high density of negative charges formed during the dissociation of carboxyl (acid) groups attracts a lot of cations, such as Na + ions, which are osmotically active and cause even more water to enter the matrix. The resulting high swelling pressure is what we call turgor. Turgor of the dermis, determined by the content and properties of HA, provides turgor .

Since the molecule contains both hydrophilic and hydrophobic regions, in solutions high-molecular HA (Mm> 1000 kDa) acquires a spatial structure in the form of a chaotically twisted ribbon, which forms a loose coil in three-dimensional space. Such coils occupy a huge volume (thousands of times larger than the volume of the macromolecules themselves!), Forming a viscous gel even at very low concentrations.

The emerging spatial networks with cells of a certain size ensure the "natural selection" of circulating molecules. Such a natural "molecular sieve" freely passes ions, sugars, amino acids, signaling molecules, but retains (and accumulates) large molecules, including various toxins.

Metabolism

HA synthesis occurs on the inner surface of the plasma membrane of fibroblasts. Monosaccharide molecules, from which the polymer chain is built, are formed from glucose; glutamine is the donor of the amino group. As the macromolecule is formed, it is excreted ( rice. 2).

Fig. 2. Synthesis of glycosaminoglycates by fibroblasts (according to H. Heine, 1997)

HA synthesis is catalyzed by the enzyme hyaluronate synthetase (HAS), which is represented by three varieties (Itano N.):

  • HASi - performs slow synthesis of chains with a molecular weight of about 200-2000 kDa,
  • HAS2 - is responsible for the rapid synthesis of high molecular weight HA with M.m. more than 2000 kDa),
  • HAS3 is the most active of the enzymes involved in the synthesis of HA from M.m. about 200-2000 kDa.

Much more hyaluronic acid is synthesized in the dermis than it is catabolized. It turns out that a significant part of it is intended for drainage through the lymphatic system, which is an important mechanism for tissue detoxification, because together with it, the "entangled" 8 molecular "networks" of exo- and endotoxins are removed. Even large HA chains with M.m. are able to penetrate into the lymphatic vessels. about 1000 kDa.

HA catabolism is of a stepwise nature, and great importance is attached to it in the regulation of the state of the matrix. Currently, the biotransformation of HA is considered as the most important factor in maintaining homeostasis and one of the universal mechanisms for the development of pathological processes (inflammation, tumor invasion and metastasis), because as the length of the initial chain decreases, fragments with their own biological activity are formed ( table 2).

HA is catabolized with the participation of hyaluronidases (types I and II), which catalyze the reactions of hydrolysis and depolymerization (extracellular degradation). Small fragments are partially phagocytosed by macrophages and undergo further catabolism with the participation of lysosomal enzymes (3-glucuronidase and (3-acetylglucosaminidase (intracellular degradation). 90% of HA that has entered the peripheral lymph flow) is destroyed in the lymph nodes, 9% and in 1% of the liver endothelial cells in the spleen.

In the body of an adult human weighing 70 kg, all organs and tissues contain about 15 g of hyaluronic acid in total, with 50% being on the skin.
Every day, about 5 g of HA is destroyed and synthesized again, that is, the "life" of this molecule is limited to several days. HA is the fastest renewable component of the extracellular matrix. For comparison: the "lifespan" of a mature collagen fiber is several months, elastin fibers generally belong to practically non-renewable structures.

Table 2. Biological functions of hyaluronic acid molecules with different molecular weights (Stern R et al, 2006)

Long chains with M.m.
about 500 kDa

They suppress angiogenesis, prevent cell migration and division, possibly due to a change in intercellular interaction, inhibit the production of the cytokine IL-1b, prostaglandin E2, and have an immunosuppressive effect.

Molecules with mass
20-100 kDa

They stimulate cell migration and division, promote wound healing, ensure the integrity of the epithelium, participate in ovulation and embryogenesis.

Short chains of mains with M.m.
less than 0.4-10 kDa

They stimulate angiogenesis, have immunomodulatory and anti-inflammatory effects.

Tetrasaccharides

They have anti-apoptotic properties, stimulate the synthesis of heat shock proteins.

HA in the life of the cell community

GC is a part of not only , but also many other organs and tissues. And at the level of the whole organism, the regulation of its biosynthesis by fibroblasts is carried out by the neuroendocrine system. An important role belongs to the hormone of the anterior pituitary gland - somatotropin, which stimulates the division and synthetic activity of connective tissue cells. Corticotropin and glucocorticoids (cortisone, hydrocortisone) inhibit the division of fibroblasts, promote their "accelerated aging", which is accompanied by a decrease in the synthesis of collagen and hyaluronic acid. Mineralocorticoids (aldosterone, deoxycorticosterone), on the other hand, stimulate the formation of HA. Estrogens have a similar effect (see Appendix "HA in the Human Body: Interesting Facts").

In the dermis, the maintenance of the HA level is provided by the mechanisms of autoregulation according to the feedback principle ( Scheme 2).

The interaction of HA with cells occurs with the participation of specific proteins - hyaladherins, which can be both elements of the receptor apparatus of cells (RHAMM, IHABP) and extracellular structures, which include verzikan, aggrecan, fibrinogen, collagen type VI (see Appendix “Interaction of HA with receptors - the mechanism for the realization of its biological activity ").

At this very place, it is probably worth stopping and thinking. What is the reason for such a wide distribution of HA in the human body? And in the animal kingdom in general? What determines the variety of mechanisms of regulation of its metabolism? Why, as degradation progresses, biological activity does not disappear, but rather changes? Summarizing all of the above and looking ahead, we can assume that the answer lies in the variety of biological functions of this unique biopolymer ( table 3).

Table 3... The biological role of hyaluronic acid

It is the basis of a hydrated extracellular matrix - a physiological environment for migration, division and differentiation of cells.

Regulates the synthetic activity of fibroblasts, including the extracellular stage of collagen synthesis.

It has an indirect immunomodulatory effect (both stimulating and suppressing the immune system).

Provides transport of nutrients and signaling molecules from blood vessels to cells, as well as excretion of waste products.

Promotes drainage and detoxification of connective tissue, is a "trap" for free radicals.

Provides tissue regeneration and damage repair (plastic function).

Participates in the regulation of angiogenesis.

Regulates tissue morphogenesis during embryonic development.

HA and aging

Whether the HA content in the skin changes with age remains controversial. However, it is known for sure that as the body ages, an increasing amount of HA passes from a free state to a bound one (with proteins). At the same time, it partially loses its unique abilities, namely: to inhibit free radical oxidation reactions, to be involved in the metabolic pathway and stimulate fibroblasts, to attract and retain water. By reducing the water content, the skin loses its elasticity, and its smooth relief is deformed by wrinkles and folds.

In cosmetology, injection procedures have the greatest success - contour plastics, biorevitalization, bioreparation. The active component of the drugs used for their implementation is hyaluronic acid (HA). Despite the ambiguous statements in the media, hyaluronic acid in cosmetology has not lost its popularity for about two decades.

The role of HA in the human body

All systems and organs are composed of cells: blood - from corpuscles, liver - from hepatocytes, the nervous system - from neurons. The space between all cells is occupied by connective tissue, which makes up about 85% of the entire organism. As a single structure, it interacts with all other tissues (epithelial, nervous, muscular, etc.) and implements their relationship with each other.

Connective tissue, depending on its composition, can be in various physical states - in liquid (blood, lymph, synovial intra-articular and cerebrospinal fluid), solid (bone), in the form of a gel (intercellular fluid and cartilage, vitreous body of the eye). It is most fully present in the skin structures - the dermis, hypodermal and basal layers.

Connective tissue is distinguished from other tissues of the body by the high development of its base with a relatively small number of cell structures. The base consists of elastin and collagen fibers, as well as complex molecular protein and amino acid compounds with amino sugars. The most important of them is hyaluronic acid.

One HA molecule is capable of binding about 500 water molecules. In a middle-aged human body, it is synthesized by fibroblasts in the amount of 15-17 grams. Half of it is contained in the cells of the stratum corneum, as well as between the fibers of elastin and collagen. It stimulates the production of these proteins, creates conditions for their fixed location, thereby giving firmness and elasticity to the skin.

Video

Aging processes of tissues

Under the influence of the enzyme hyaluronidase, hyaluronic acid is destroyed. The processes of its restoration and splitting are going on continuously. About 70% is destroyed and restored within a day. The prevalence of a particular process depends on:

  • daily and seasonal biorhythms;
  • age;
  • psychological state;
  • poor nutrition;
  • nicotine intoxication and excessive UV exposure;
  • taking certain medications, etc.

These factors affect not only the synthesis of HA (hyaluronate), but also its structure. A decrease in its amount leads to a decrease in the bound water in the tissues and the appearance of signs of aging. Defective molecules retain the ability to bind water, but lose the ability to give it away. In addition, natural age-related processes lead to the concentration of HA in the deep skin layers, which is the cause of intercellular tissue edema at the border of the dermis and hypodermis and dehydration of the more superficial layers.

All these processes increase with age and under the influence of negative factors and lead to dry skin with simultaneous puffiness of the face and edema under the eyes, a decrease in its elasticity and firmness, the appearance of wrinkles and pigmentation.

Types of HA in the body

Its uniqueness lies in the presence of molecules with different polysaccharide chain lengths. The properties of hyaluronic acid and its effect on cells largely depend on the length of the chain:

  1. Short-chain molecules, or low molecular weight hyaluronic acid, have anti-inflammatory effects. This type of acid is used to treat burns, trophic ulcers, acne, psoriasis and herpetic eruptions. It is used in cosmetology in the form of one of the components of tonics and creams for external use, since without losing its properties, it penetrates deeply into the skin for a long time.
  2. Medium molecular weight HA, which has the property of suppressing migration, cell multiplication, etc. It is used in the treatment of eyes and some types of arthritis.
  3. High molecular weight - stimulates cellular processes in the skin and has the property of retaining a large number of water molecules. It gives the skin elasticity and high resistance to external negative factors. This type is used in ophthalmology, surgery, and in cosmetology - in preparations for injection techniques.

Industrial types

Depending on the production technology, sodium hyaluronate is divided into two types:

  1. For a long time, preparations with hyaluronic acid of animal origin have been used. It was obtained by enzymatic cleavage of crushed parts of animals (eyes and cartilage of cattle, cockscombs, synovial intra-articular fluid, umbilical cords) as a result of a special two-stage purification and sedimentation. The technology involved the use of distilled water and high temperature (85-100 degrees). A significant part of the high-molecular-weight fraction was destroyed, turning into a low-molecular one. In addition, proteins of animal origin remained.

    The effect after injections of such drugs for the purpose of cosmetic face correction did not last long, sometimes it contributed to the formation of dermal nodes. But the drug was especially dangerous because it often became the cause of severe inflammatory and allergic reactions due to the presence of animal protein. Therefore, this technology is almost never used.

  2. Recently, in the pharmaceutical industry, HA is obtained by the method of biotechnological synthesis. For these purposes, microorganisms (streptococci) grown in wheat broth are used. They produce hyaluronic acid, which is purified, dried and subjected to repeated bacteriological and chemical studies in subsequent stages. Such a drug almost completely corresponds to the acid produced in the human body. It almost does not cause allergic and inflammatory reactions.

Application in cosmetology

Hyaluronic acid is applied to the skin and subcutaneous layers in a variety of ways:

  1. Injection.
  2. No injection.

Injection procedures with hyaluronic acid are used in such techniques as:

  • , and - the introduction of the drug into the middle layers of the skin; it is used for age-related changes, dry skin and to increase its elasticity, tone and color, eliminate acne, stretch marks, etc.; the duration of the preservation of hyaluronic acid in the dermis - up to 14 days;
  • - filling the subcutaneous structures with the substance in order to smooth out wrinkles and correct the contours of the face; the drug remains under the skin for 1-2 weeks;
  • and - administration of modified hyaluronic acid, which remains in the skin for up to 3 weeks.

Questions

Which is Better: Botox or HA?

Given the multidirectional mechanisms of action of botox and hyaluronic acid, they are used to achieve various effects. A combination of them is possible. However, it must be remembered that at least two weeks must elapse after the introduction.

Is it possible to combine the administration of collagen fillers and HA?

Collagen and HA fillers work well together. The first provides the skin with density and structure and lasts an average of 4 months, the second - natural moisture and strength for 6-9 months.

Any use of hyaluronic acid injections should only be carried out by a cosmetologist.

Hyaluronic acid was discovered in 1934, the first detailed studies of it began in 1949 - 1950. This substance was isolated from various animal tissues - joint fluid, umbilical cord and cock's crest tissues. In addition, in 1937, hyaluronic acid was obtained from streptococcal capsules. The first studies of the physical and chemical properties of hyaluronic acid were carried out by X-ray crystallography.

Problems of obtaining GC

The main problem in the study of hyaluronic acid, which the scientists faced, was the difficulty of isolating it in its pure form, purified from proteins and other components. The difficulty arose because there was always a risk of destruction of the polymer structure of hyaluronic acid during the cleaning process. At the same time, scientists have tried a variety of methods of physical, chemical and enzymatic cleaning.

A little later, studies began on the possibility of biosynthesis of hyaluronic acid. In 1955, this method was first found. A group of scientists isolated hyaluronic acid molecules from streptococcal extract. Thanks to this discovery, it became possible to synthesize hyaluronic acid - using an enzymatic fraction taken from streptococci.

Hyaluronic Acid - Application

The main breakthrough in the use of hyaluronic acid occurred in the 50s. Thanks to the discovery of this substance for use in medicine, its industrial production and popularization as a medicine began.

In 1970, hyaluronic acid was approved as a proven effective remedy for arthritis - after receiving positive results in animal testing. As a result of the experiment, a pronounced clinical effect with a decrease in symptoms was noted.

A few years later, hyaluronic acid began to be used in implantable intraocular lenses, which quickly made it one of the most commonly used components in surgical ophthalmology. From that moment on, various methods and fields of application of hyaluronic acid began to be proposed and tested.

GK today

In the 90s hyaluronic acid found wide application in aesthetic medicine and cosmetology, due to its unique moisture-retaining, antiseptic and antioxidant properties. It is still used for various cosmetic purposes, and research on its properties and possible applications continues.

Today, references to hyaluronic acid are full of both glossy publications and the pages of ordinary media. Over the past few years, we have not ceased to repeat that “the secret of eternal youth of the skin has been revealed” and they offer to use this “elixir”. Let's try to figure out what is more in this unhealthy excitement - truthful information, accurate commercial calculation or banal philistine delusions.

Discoveries of the past that did not meet expectations

If you look into the very recent past, you can remember that similar situations have already been in the history of medicine:

  • The discovery of penicillin was presented as a complete victory over microorganisms (which, unfortunately, did not happen, despite the current spectrum).
  • The insulin produced was predicted to be victorious over (a vital and essential drug for diabetics, but a complete victory over diabetes is still a long way off).
  • The use of the first antipsychotics was presented as a cure for certain mental disorders, but even here everything is far from ideal expectations.

In general, the true picture after some time still differs from the forecasts and initial estimates. Therefore, it is very important to treat everything critically and as objectively as possible.

Debunking the myths about hyaluronic acid

None of the doctors will argue that hyaluronic acid is important for the human body, but that much information that can be found in the media today and which is presented as true, alas, does not come to us from professionals. Most often, innovative ideas are brought to the people by various beauty experts, self-taught bloggers and other people without specialized medical, pharmaceutical or biological education. They speak about a medicine based on their own evaluative impressions, information from questionable sources, or information taken out of context

This is how delusion is born. Let's try to separate the wheat from the chaff and understand this issue in more detail.

True

The main misconception is that the drug is called in the singular, but it is correct to call it in the plural - acids, since this is one of the compounds of the group of acidic mucopolysaccharides, which includes other compounds of similar composition and properties, and their mass can vary widely. Since the overwhelming majority of drugs released under the name "hyaluronic acid" are made from biological raw materials without special separation of fractions, it is completely incorrect to consider the drug as one, pure, compound.

Hyaluronic acid is the result of discoveries in beauty laboratories over the past two to three decades.

The substance itself was discovered back in 1930, and the study of its properties, functions, as well as the possibilities of application were taken up almost immediately after the discovery. The research itself did not stop, and starting from the 70s of the last century, their intensity began to increase.

This substance is used in cosmetic and cosmetic products

In addition to this developed area, hyaluronic acid is used for various diseases of other organs and systems as a medicine.

In cosmetic products, it improves the penetration of nutrients into the skin

Does not affect the level of cellular and intercellular permeability for various substances

Aging of the skin is associated with the loss of fluid due to a decrease in the level of substances of this group in all layers of the skin

If the decrease in the content of hyaluronates occurs with age, it is not so significant, and aging, including skin aging, is a complex multifaceted general biological process and it is simply stupid to reduce its manifestations to such banal reasons

The truth about hyaluronic acid

All properties and characteristics and distinctive features of hyaluronic acid are described in detail in the scientific and medical literature. However, it is oversaturated with many terms, which makes the available information not always understandable for a common layman.

If we try to simplify everything a little, it turns out that:


Each of the factions has its own set of properties and characteristics. So low molecular weight varieties substances have an excellent anti-inflammatory effect, which ensured their use for burns, trophic ulcers, herpetic eruptions, psoriasis ... Medium molecular weight hyaluronic acid able to suppress cell multiplication and migration. Due to these properties, it is used in the treatment of some arthritis and eye diseases. High molecular weight fractions keep a huge number of water molecules around them and stimulate cellular processes in the skin itself. This type of hyaluronic acid has found its application in surgery, ophthalmology and cosmetology.

It's important to know! It is categorically impossible to use a drug with an unspecified size of the molecules of the active substance, since it is possible not only not to achieve the desired result, but also to worsen the condition.

The main indications for the use of hyaluronic acid

It should always be remembered that the introduction of drugs into the body hyaluronic acid injection is primarily a medical manipulation. There are quite strict medical criteria for the use of various techniques and procedures.

So, the main indications for the use of hyaluronic acid are:

  • the appearance of wrinkles (reduced skin turgor) due to loss of moisture;
  • an increase in the severity of existing wrinkles;
  • pronounced mimic wrinkles;
  • the need to normalize the skin relief;
  • the need to improve the turgor and contour of the red border of the lips.

Hyaluronic acid preparations in aesthetic medicine

In modern cosmetology, the demand for hyaluronic acid in the form of injections or other forms of the drug is explained by:


The modern pharmacological market offers hyaluronic acid in the form of injections. Moreover, it can be in the form:

  • Meso cocktail, which includes the main substance, supplemented with panthenol, vitamins, coenzymes, cell growth factors, peptides, etc.
  • Fillers- a dermal filler made from cross-linked HA, which biodegrades over time - it is absorbed in the body. It is produced in the form of a gel of various viscosities. The more viscous the substance, the more problems it is designed to cope with.
  • Redermalizants and biorevitalizants... Currently, 3 generations of these drugs can be found on the shelves of pharmacies. The latter are based on nucleic acids that create complexes with HA that can restore cell DNA and accelerate the production of their own hyaluronic acid, as well as elastin and collagen.
  • Bioreparants- preparations containing altered HA, to the chain of which peptides, vitamins, amino acids are attached. They have a prolonged and enhanced action.

Note: in the beauty industry, ointments, creams, gels, lotions for external use can be used, but their effectiveness is much lower than that of hyaluronic acid for injection.

The main types of procedures to improve the condition of the facial skin

The most popular injection procedures with hyaluronic acid are:


The main contraindications to the use of hyaluronic acid

If marketers are trying to assure you that hyaluronic acid injections, wherever they are carried out, are as safe as possible, know: this is a lie! Against the background of certain procedures, they are really safer, however, this drug has its own contraindications.

The main ones include:

  1. Any allergic reaction to the active substance or its components.
  2. Any infectious diseases in the acute period.
  3. Pregnancy, childbirth and subsequent lactation.
  4. Connective tissue pathology.
  5. General and systemic diseases, such as autoimmune lesions, oncological pathology of any organs and systems, sugar, pathology of the blood coagulation system.

In addition, birthmarks, moles, scars and inflammation should not be located at the injection site. If these contraindications are not followed, the results can be disastrous.

The effectiveness of hyaluronic acid creams

A separate group of drugs, and quite common, are creams with hyaluronic acid. They are applied by being applied to the surface of the skin, where they have a direct effect.

For superficial changes, skin protection, products containing high molecular weight fractions are used, which create a protective layer and do not penetrate into the skin.

To correct deep, age-related changes, funds with low-molecular fractions of the active substance are better suited, since it can partially penetrate to a certain depth into the inner layers, where their biological action is carried out.

In recent years, non-injection techniques have become increasingly popular, implying the application of a gel to the skin, followed by exposure to microcurrents, a laser, and ultrasound.

I would like to end with some advice: everything has its own time and reasons, and the basic rule of a healthy life, good mood and good looks is moderation. In pursuit of beauty, try to use even a product such as hyaluronic acid, no frills, and your skin will look good even in old age.

You will receive more detailed information on the use of hyaluronic acid preparations for the face by watching the video review:

Elena Nikolaevna Sovinskaya, therapist.