Abstract

One of the most common questions aesthetic clients ask is “How do radio-frequency energy treatments work to tighten skin?” In this article, industry experts from Medisico PLC explain the biological mechanism of how heat shock proteins revitalise skin after radio-frequency energy treatments. When tissue is heated or stressed, as occurs after radio frequency energy treatments, cells naturally begin to produce tiny proteins that stabilise the cell. One of the roles of heat shock proteins is to help newly formed or improperly folded proteins to fold in to their correct shape, because shape is vital to function. For example, collagen is strong because it is actually three strands of pro-collagen bound together, and the helper protein that holds the strands in alignment as they bond is a heat shock protein. This paper discusses the various types of heat shock proteins and their roles in tissue regeneration after radio-frequency therapy.

Introduction

In order to educate and advise clients about aesthetic techniques, it is helpful to be familiar with the mechanisms of tissue regeneration. Radio-frequency energy treatments are a popular option for clients interested in non-surgical methods of reducing tissue laxity. These treatments initiate a process of dermal tissue remodeling by stimulating fibroblasts to proliferate and produce new collagen and heat shock proteins (Touma and Gilchrest, 2003).When the first heat shock proteins were discovered, they were noted to be over-expressed by cells that had been exposed to heat (Ritossa, 1962). We now know that there are several sizes of heat shock protein that play many different roles in the cell. In fact, we’re still discovering all of the things heat shock proteins can do. So far, we know how heat shock proteins repair damaged proteins, aid the cells to synthesise new proteins, help assemble proteins that are newly made, and promote the survival and proliferation of cells under stress.

I. Types of Heat Shock Proteins

The heat shock proteins are actually a family of proteins, classified according to their molecular size as measured in kilo Daltons (a Dalton is the number of grams per mole of asubstance.) Here, we discuss some of the sizes that are particularly relevant to dermal remodeling. The sizes range from HSP27, which is the is the smallest at 27 kD, to HSP70,which is 70 kD. For comparison, Type 1 collagen is about 130 kDa. Helping proteins fold is called molecular chaperone function, and all of the members of the heat shock protein family do this. In addition, each size of HSP has been found to have particular specialties.

HSP27 promotes cell survival and works to contract newly formed tissue scaffolding, which provides a tightening and strengthening effect. HSP47 is known as the collagen specific chaperone, since it stabilises pro collagen filaments in the correct conformation to make collagen chains (Nagata et al., 1988). The HSP70 class binds and folds a broad array of proteins, increases cell survival (Nonaka et al., 2004), and helps determine whether a protein should be flagged for repair or sent to the recycling bin (Wagstaff et al., 2007).

II. How Radio-frequency Treatment Activates Heat Shock Proteins

In order to study the time course of heat shock protein expression after radio-frequency therapy, one investigator recruited 22 clients undergoing elective surgical face lift or
abdominoplasty. After treatment with a bipolar radio-frequency device, they found that,
HSP70 expression peaks at an early time point and diminishes rapidly after two days. Next, the collagen chaperone HSP47 increases progressively over the course of 10 weeks. Researchers noted significant increases in connective tissue components includingtropoelastin, fibrillin, and proc ollagen 1 and 3 as early as 28 days after treatment (Hantash etal., 2009). Aesthetic practitioners know that radio-frequency treatments can take several weeks to achieve the desired effect, and part of the reason is the timeline of there generative process taking place.

III. Heat Shock Proteins and Fibroblast Growth

Fibroblasts are skin cells that maintain the structural framework of the skin by generating collagen and extracellular matrix. Fibroblasts also make heat shock proteins when they are stressed, and the presence of heat shock proteins actually stimulates fibroblasts to replicate(Capon and Mordon, 2003). Heat shock proteins do this by causing the cell to produce a tissue growth factor called transforming growth factor (Cao et al., 1999). TGF-βis also produced by inflammatory cells, including neutrophils and macrophages (Steenfos, 1994) and influences the proliferation, differentiation and motility of fibroblasts. TGF-βnormally activates HSP27by phosphorylation, leading to further production of connective tissue growth factor and type1 collagen (Lopes et al., 2009). TGF-βalso ramps up the production of more heat shock protein (Sasaki et al., 2002). When TGF-βlevels are reduced experimentally, it blocks the ability of fibroblasts to produce connective tissue (Mori et al., 2004).

IV. HSPs Promote Cell Survival

Two of the heat shock proteins, HSP70 and HSP27 are able to directly prevent programmed cell death, or apoptosis. When cells are sufficiently stressed, the cell may begin a process of breaking itself down before bursting. This auto-digestion process makes it easier for phagocytotic cells to clear away the cell remnants, called apoptotic bodies. HSP27 is normally found in the cell bound to actin, which forms part of the cytoskeletal scaffold and maintains the shape of the cell. During cell stress, HSP27 detaches from actin. HSP70 andHSP27 both migrate from the cytoplasm and into the nucleus to stop cell death (Nahomi etal., 2014). HSP70 binds to the caspase binding domain of apoptotic protease activating factor1 (APAF-1) to block cell death signaling (Saleh et al., 2000). HSP27 phosphorylates AKT in order to down regulate the apoptosis process(Qi et al., 2014).

V. Heat Shock Proteins HSP27, HSP47 and HSP70 are Key for Tissue Regeneration

One study that was particularly informative regarding the way heat shock proteins are important to collagen deposition focused on keloid scars. Keloid scars are an example of a disregulated collagen synthesis process that overproduces collagen, resulting in a hypertrophic growth of scar tissue. Normal and keloid tissue was collected from patients undergoing keloid scar revision, and found that in keloid tissue HSP70, HSP47, and HSP27 were over-expressed. In comparison, two other sizes of heat shock protein, HSP60 and HSP90, were not increased in the keloid scar. HSP60 and 90 are thought to have different roles in the cell unrelated to tissue matrix regeneration. HSP60 is responsible for mitochondrial processes and HSP90 works with cell signalling molecules such as protein kinases and steroid receptors (Totan et al., 2011).

VI. HSP47 is Collagen Specific

To examine the role of HSP47 in collagen synthesis, a study by Ohba et al used rats undergoing wound healing. HSP47 production was eliminated using an antisense oligonucleotide to bind HSP47 RNA and prevent it from being transcribed into protein. When HSP47 was eliminated, western blot analysis determined that collagen precursor proteins (procollagen types 1 and 3) were no longer synthesised for wound repair. Using RT-PCR, they determined that the mRNA for pro collagen was still being manufactured, but the code wasn’t being used to produce a protein. Thus, disrupting HSP47 prevented collagen production at the translation step, in which proteins are built from mRNA. This finding indicated that HSP47 is required not just for assembly of collagen, but also in an earlier step during the translation of procollagen (Radio-frequency energy treatments to tighten skin rely on heat shock proteins to achieve atissue tightening effect. In particular, the heat shock proteins HSP27, HSP47, and HSP70 arethe sizes of heat shock protein involved in collagen matrix regeneration. Heat shock proteinspromote the survival and replication of skin fibroblasts, and support the production andassembly of structural components such as collagen. The timeframe of heat shock proteinexpression after radio-frequency treatment explains why clinical results develop over thecourse of several weeks.Ohba et al., 2003).

VII. HSP27 Governs Tissue Matrix Contraction

HSP27 is activated by TGF-β, and similar to HSP47, blocking HSP27 activation prevents the production of collagen and growth factor (Lopes et al., 2009). Following collagen deposition, contraction of the collagen matrix is another key process that occurs after radio-frequency treatment. When fibroblast cells were placed in an artificial matrix of collagen and stimulated using platelet-derived growth factor and lisophosphatidic acid, they begin to contract the matrix in order to strengthen it. Cells that had high levels of HSP27 contracted the matrix more than cells that expressed low levels of HSP27. Fibroblasts over-expressing HSP27 were also better at migration and adhesion. This finding implicates HSP27 to be extremely important to the desired skin tightening effect of aesthetic RF therapy (Hirano etal., 2004).

Conclusion

Radio-frequency energy treatments to tighten skin rely on heat shock proteins to achieve a tissue tightening effect. In particular, the heat shock proteins HSP27, HSP47, and HSP70 are the sizes of heat shock protein involved in collagen matrix regeneration. Heat shock proteins promote the survival and replication of skin fibroblasts, and support the production and assembly of structural components such as collagen. The time frame of heat shock protein expression after radio-frequency treatment explains why clinical results develop over the course of several weeks.

Key points

• Heat shock proteins play a variety of roles in tissue rejuvenation after radio-frequency energy treatments.
• Heat shock proteins promote fibroblast proliferation through the growth factor TGF-β.
• HSP47 is the collagen specific protein, and helps assemble collagen from individual strands
of procollagen.
• HSP47 and HSP27 are required for collagen formation.
• HSP27 increases for several weeks after RF therapy, and helps the newly formed tissue matrix to tighten.

Key words:

Heat Shock Proteins, Radio-frequency, collagen, tissue regeneration, healing mechanisms.

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Conflict of interest statement:

The authors of this article are employees of Medisico PLC and
have no conflict of interest with the research reported in this review.