Kazal Boron Biswas, Yuka Kawai, Kyoko Kanai, Kotaro Sakamoto, Teruaki Masutani, Arunasiri Iddamalgoda • Ichimaru Pharcos

Hyaluronic acid (HA) is the simplest form of the glycosaminoglycans in nature and is composed of thousands of repeating alternative units of glucuronic acid and N-acetylglucosamine.¹ It is quite a simple but big molecule, and the molecular weight sometimes reaches several million Dalton. HA is extremely hygroscopic, meaning that it may contain up to 1000-fold more water than its own molecular weight. It is ubiquitously present in humans and animals as a major constituent. Although many organs maintain high concentrations of HA, skin contains approximately half of all HA in the body.² It is produced at the plasma membrane by an enzyme, hyaluronic acid synthase. The important issue is that the half-life of HA is very short. In fact, approximately one-third of the body’s total HA is turned over daily. Therefore, precise control of HA degradation and synthesis is required for this turnover. Due to the huge impact of HA on skin, it is widely used in cosmetics nowadays in various forms such as hydrogel, dermal filler, intradermal injection, creams and so on.³

HA Function in Skin

HA is ubiquitously present in both the epidermis and dermis of human skin, and it plays an important role in maintaining total skin homeostasis. The main function of HA in the epidermis is to regulate keratinocyte cell behavior, including proliferation and differentiation, as well as maintain moisture by retaining water as it is known as one of the most important components of Natural Moisturizing Factor (NMF). 

On the other hand, HA is considered as one of the most important components of the extracellular matrix (ECM) of dermis, and several biological activities have been identified in the dermis despite the simplicity of its structure. It can retain a lot of water and maintains dermal moisture. By holding water, it functions not only as a biological glue that participates in the lubrication and viscosity of tissues, but also maintains a micro-environment which is important for nurturing fibroblast’s health through passage of nutrients. Moreover, the dermis is generally thicker (about 10-fold) than the epidermis;⁴ i.e., there are lot of empty spaces in the dermis. HA gel-like material plays an important role in filling up those empty spaces.⁵

HA Function in Dermis

Besides all these functions of HA, it has another important function which is not as well-known as it should be, and that is the maintenance of ECM structure on top of its water retention characteristics. In fact, ECM structure is an integrated network of many factors such as collagen, elastin, HA, proteoglycan, etc., and a comprehensive contribution from each component is necessary to hold the structure properly. 

If we think about individual contribution of these components, we can say that the primary contribution comes from the HA itself because, for disintegration of the total ECM structure, it is necessary for the HA to be degraded first before the degradation of collagen and elastin molecules.⁵ We also prepared in our laboratory a representative structure of collagen gel in the presence and absence of HA followed by its disintegration for a certain time. It was found that collagen alone is not enough to stabilize the structure, HA also is equally important. The disintegration of collagen-only gel appears faster than that of collagen-HA gel (Fig. 1).

It is well-accepted that the degradation of ECM infrastructure leads to wrinkling and sagging. People generally know that collagen and elastin are the main or only components responsible for maintaining the structure of ECM, and nobody usually talks about the importance of HA in terms of its role in structure formation. Recently, it has been reported that the loss of HA results in wrinkling and sagging, which are inversely proportional to the amount of HA.⁶ Moreover, HA has been reported to protect collagen and elastin by inhibiting the entry of their degrading enzymes into the initial network made by HA itself and in conjunction with various proteoglycans.⁷ Therefore, it is reasonable to think that HA provides more supports to the skin compared to other matrix components in the dermis. Another important point is that the half-life of HA is only 1-1.5 days, whereas elastic fibers and collagen fibers have half-lives of years. Hence, it appears very important that we need to take care of HA critically.

One may think that it is possible to produce HA from within as there is HA synthesizing machinery in the skin. But it is important to keep in mind that, with aging, HA degrading enzymes become more prominent than the HA synthesizing enzymes creating an imbalance toward more degradation than production. It has already been reported that there is an age-dependent gradual loss of HA in human skin.⁸ Photoaging also reduces HA levels.⁶ Therefore, it is better to think about the protection of HA from inside rather than thinking about its production or injection because of the higher degradation rate of HA during intrinsic and extrinsic skin aging.

Mechanism of Degradation

Conventional mechanisms of HA degradation involve the hyaluronidase (HYAL) enzymes, mainly HYAL1 and HYAL2, which depolymerize the large HA into individual sugar units following lysosomal action. These free sugar units then extrude into the cytoplasm and are used up by the cells for different biological reactions.¹ Now, a new mechanism of HA degradation system has recently been proposed, which is independent of HYAL system. This novel system involves a protein called HYBID (HYaluronan-Binding protein Involved in hyaluronan Depolymerization) alias KIAA1199 which binds and degrades high molecular size (>1000 kDa) HA into intermediate size fragments with molecular weights ranging from 10 kDa to 100 kDa (Fig. 2).⁹ These intermediate size fragments are released into the extracellular space and are known to cause harmful effects including inflammation and pain in various tissues, but their effects in skin fibroblasts have not yet been determined. These authors have also demonstrated that it is not the HYAL enzyme, but HYBID is responsible for the degradation of HA in human skin fibroblasts.

HYBID is generally expressed in many cell types such as astrocytes,¹⁰ fibroblasts,⁹ osteoblasts¹¹ and various cancer cells.^12,13 The regulation of HYBID expression in skin fibroblasts has been elucidated using many factors including cytokines and chemokines.⁹ It has been demonstrated that its expression is significantly up-regulated by the treatment with histamine, a well-known factor usually released from mast cells upon UV irradiation. Recently, it has been reported that the expression of HYBID is increased in photoaged skin, and this increase is probably due to the increased release of histamine from mast cells.⁶ It has also been revealed that HYBID expression is negatively correlated with the levels of HA, and positively correlated with sagging and wrinkle in photoaged skin, suggesting that HYBID-mediated HA reduction in the dermis contributes to the development of wrinkle and sagging, at least, in this type of skin.⁶ In this study, we have revealed that HYBID expression is higher in adult fibroblasts (Fig. 3A) compared to newborn fibroblasts implying that its level is increased with natural aging.

Inhibition of Degradation

As HYBID has been established as a novel and innovative factor for the degradation of HA, the inhibition of this factor is important to protect the HA from loss. Ichimaru Pharcos has developed a new active ingredient, Artemisia Capillaris Flower Extract (ACFE), which is extracted from a medicinal plant, Artemisia capillaris Thunb. (Compositae). ACFE inhibits the mRNA expression of HYBID not only in adult fibroblasts but also dose-dependently in histamine-induced newborn fibroblasts (Fig. 3A, B). Since HYBID is directly involved in the degradation of HA, we also checked the effect of ACFE on the protection of large HA. After the newborn fibroblasts were treated with ACFE for around three hours, they were exposed with a HYBID inducer, histamine, for an additional three hours, followed by the addition of fluorescein isothiocyanate (FITC)-labeled large HA (1,200-1,600 kDa). 

After 48 hours of incubation, it was found that not only the large HA decreases, but also the small size HA increases in the medium by the effect of histamine-induced HYBID. In contrast, the use of ACFE dose-dependently reversed the effect, which leads to the protection of large HA from degradation (Fig. 3C). In adult fibroblasts, too, ACFE suppresses the degradation of large HA (data not shown). In fact, both in histamine-stimulated newborn fibroblasts and non-stimulated adult fibroblasts, large HA increases and small HA decreases by the effect of ACFE, and it is thought that this effect is achieved through the inhibition of HYBID. Therefore, ACFE is expected to protect the skin from both extrinsic and intrinsic aging.

Clinical Aspects

As HYBID is responsible for the degradation of HA, which is one of the key components of dermal matrix, the efficacy of ACFE has been tested against wrinkle and sagging in a human clinical study. The investigation was carried out in a total of 13 subjects (male and female, average age: 43 years) for a period of four weeks. The one half of the face of each participant served as control (placebo) and the other half of the face served as test (lotion containing 1% ACFE). For measurement of sagging, an adhesive tape with a circular hole was fixed on the cheek and then a certain weight-load was set with a string with one end holding the weight and other end holding the circular hole. The weight was determined in advance in such a way that the cheeks would move downward by 1-2mm due to gravity. The actual distance travelled by the cheeks due to gravity was measured by Visia Evolution (Canfield Scientific) before and after the weight load in the absence or presence of ACFE at 0- and 4-weeks. The method of measuring sagging distance is shown schematically (Fig. 4A). Antera 3D (Miravex) was used for wrinkle measurement and wrinkle imaging.

The ACFE formulation has been shown to decrease sagging significantly (p<0.05) when compared between week-0 (baseline) and week-4 (Fig. 4B). Moreover, ACFE-formulated lotion decreases significantly (<0.05) the extent of sagging after four weeks (Fig. 4C), when compared with control. Regarding wrinkles, the ACFE formulation, compared to placebo formulation, significantly reduced (p<0.05) the maximum depth of eyelid wrinkle after four weeks (Fig. 4D, E).

Discussion & Conclusion

ACFE has the potential to inhibit histamine-induced HYBID expression in human skin fibroblasts in culture (Fig. 3B). This inhibition might have led to the decreased degradation of HA in vitro (Fig. 3C). Histamine is a released factor from mast cells under UV irradiation, and HYBID expression has been reported to be increased in photoaged skin.⁶ There is a possibility that histamine is released due to extrinsic aging of skin and causes the degradation of HA by inducing the expression of HYBID. 

In this aspect, ACFE has strong potential to inhibit extrinsic aging. ACFE decreases the expression of HYBID as well as the degradation of large HA in adult fibroblasts (Fig. 3A) meaning that ACFE may also be involved in inhibiting intrinsic aging of skin. Furthermore, we show that this product decreases the extent of wrinkle (Fig. 4D, E) and sagging in clinical study (Fig. 4A-C). It has been reported that there are strong correlations between HYBID-mediated HA degradation and the indices of wrinkle and sagging.⁶ Our data implies that the effect of ACFE on the decrease of wrinkle and sagging in a human clinical study might be provided through the inhibition of HYBID-mediated HA degradation. In conclusion, it is suggested that HA with its intact size provides strong structural support against aging. We have proposed a specific plant sourced ingredient, ACFE, with interesting properties for skin aging care. Thanks to ACFE which is capable of taking care of both extrinsic and intrinsic skin aging by suppressing the novel mechanism involving HYBID, and simultaneously by boosting our system’s own HA through its protection.

More info: Ichimaru Pharcos, www.ichimaru.co.jp/english/

References

1. Gupta RC et al. Front. Vet. Sci., 6:192 (2019).
2. Meyer LJ et al. J. Invest. Dermatol., 102: 38538 (1994).
3. Bukhari SNA et al. Int. J. Biol. Macromol., 120: 1682 (2018).
4. Leider M. J. Invest. Dermatol., 187 (1948).
5. Nagaoka A et al. J. Biol. Chem., 290: 30910 (2015).
6. Yoshida H et al. Br. J. Dermatol., 179: 136 (2018).
7. Yoshida H et al. Int. J. Mol. Sci., 20: 5804 (2019).
8. Longas MO. Carbohydr. Res., 159: 127 (1987).
9. Yoshida H et al. Proc. Natl. Acad. Sci. USA, 110: 5612 (2013).
10. Marella M et al. Glycobiology, 28: 958 (2018).
11. Chen L et al. Cell Death Dis., 10: 126 (2019).
12. Zhang Y et al. Oncol. Rep., 31: 1503 (2014).
13. Liu J et al. Biomed. Pharmacother., 138: 111507 (2021).