Molecular Mechanisms Involved in Hair-Follicle Response to the Laser

The molecular mechanisms involved in laser-mediated hair removal still remain obscure. It is well known that the response of cells to increased temperature is accompanied by elevated expression of superfamily of proteins termed “heat shock proteins” (HSP). Each

subfamily of HSPs is composed of members expressed either constitutively or regulated inducibly. HSPs participate in essential physiological processes, such as regulation of cell cycle, differentiation, and programmed cell death. They are also called “stress proteins” because their synthesis is stimulated by variety of stresses including heat and irradiation. The HSPs induced in response to stress have a substantial role in cell protection against death. The HSPs induced by heat bind to denatured proteins to restore their structure or to bring them to degradation pathways. In addition, stress activated HSPs are able to control several intracellular apoptotic events [65,66]. It has been shown that 815-nm diode laser induces long-lasting expression of 72-kDa heat shock protein in normal rat skin [67].

In experiments carried out in our laboratory we observed that exposure of hair follicles to diode laser irradiation-induced synthesis of HSP in the follicular cells (Fig. 9.3). Shortly after irradiation up-regulated expression of HSP70 was detected in the follicular outer and inner root sheaths, as well as in the dermal papilla. In addition, an appearance of HSP70 expression was detected in the melanogenic area above the dermal papilla, which is in line with the concept that hair follicle melanocytes are the major target for the laser. However, the progressive decrease in HSP70 expression in the hair follicles over time was also seen, suggesting activation of cell death in this area. Thus, our data together with literature reports suggests that enhanced expression of HSP after laser irradiation reflects a physiological response of the hair follicles to irradiation stress, and it might serve as a first protective mechanism against laser-induced cell death. Therefore, we hypothesized that HSP inhibi­tor application prior to laser treatment may result in decrease of hair follicle resistance to apoptosis. In addition, HSP inhibitors may allow a decreasing the dose of laser irradiation to achieve hair growth inhibitory effects. We tested this hypothesis by cotreatment of the hair follicles with HSP inhibitor KNK437 and diode laser, using the isolated hair follicle ex vivo model. Our data suggest that KNK437 and laser irradiation have a synergistic effect on reduction hair growth in vitro (Fig. 9.3) [68].

Cellular damage caused by the laser could also be accompanied by an increase in p53 expression. p53 is a transcription factor that mediates cellular apoptosis in response to the variety of stresses, including ionizing irradiation, chemotherapy, and exposure to a thermal insults [69-71]. However, in the absence of p53, stressful stimuli cause cell necrosis [72]. Topping et al. observed that a single exposure to 15 J/cm2 ruby laser caused an appearance of p53 expression in epithelial cells lining the hair follicles with the damaged hair shafts. p53 expression was found to be extended in a radial fashion within the hair follicle. The more severely damaged cells were incapable of expressing p53, most likely because of their death. In hair follicles that reached the highest temperature, p53 expression was also seen in the sebaceous glands [13].

In our laboratory, we explored the effect of laser treatment on p53 expression, catagen induction and growth reduction using isolated human hair follicles. p53 expression was examined four hours after direct exposure of hair bulbs of isolated anagen hair follicles to a diode laser dose that induced catagen formation (2 W for 100 ms) and the dose that reduced hair growth without inducing catagen but causing hair follicle destruction (4 W for 50 ms) (Fig. 9.4). The hair follicles treated with 2W for 100 ms laser showed substantial up-regulation in p53 expression in the outer root sheath, hair matrix, and in the dermal papilla, compared to the untreated hair follicles. In contrast, the hair follicles treated with 4W for 50 ms laser showed a complete absence of p53 immunoreactivity in parts of hair follicle exposed directly to the laser and presumed to reach coagulative temperature levels,

while p53 immunoreactivity was still present in the distal parts of the outer and inner root sheaths where temperature had not reached the threshold level for severe damage. This data is in agreement with observations from Topping et al. described earlier [13], and suggests that moderate laser fluences activate pro-apoptotic pathways and p53-dependent cellular changes in the hair follicles, whereas high laser fluences cause severe damage to the hair follicle resulting in its necrotic death.

In addition to p53 protein, another molecule that may regulate cellular apoptotic events is the protein Bcl-2. In our laboratory, we investigated the expression of this anti-apoptotic pro­tein in the hair follicles in response to varying diode laser fluences. Bcl-2 is a mitochondrial – anchored protein, which forms ion channels capable of maintaining membrane homeostasis

and preventing cytochrome c release, thus enhancing cell survival [74]. Bcl-2 protects cells against apoptosis via suppression of cysteine proteases called caspases, which are part of the proteolytic caspase cascade that is activated by diverse apoptotic stimuli [75]. It was shown that induction of apoptosis by heat and gamma-radiation in a human lymphoid cell line was accompanied by a decline in Bcl-2 protein levels and was mainly executed in a caspase-de- pendent pathway [76]. In the hair follicle, Bcl-2 is prominently expressed in the follicular dermal papilla throughout the hair cycle, which demonstrates a high anti-apoptotic potential of this hair follicle compartment [34]. In our studies, we also detected a prominent Bcl-2 immunoreactivity in the dermal papilla and hair matrix keratinocytes in control hair follicles. However, the hair follicles treated with 2W laser at 100 ms pulse showed a significant decrease in Bcl-2 immunoreactivity in the dermal papilla, as well as in the hair matrix cells. A com­plete absence of Bcl-2 expression was seen in hair follicles treated with 4W laser at 50 ms pulse. The elevated expression of Bcl-2 in a follicular papilla at the low laser power suggests activation of anti-apoptotic response in the hair follicle and the viability of dermal papilla cells, while the absence of Bcl-2 in hair follicles exposure to the higher laser power indicates occurrence of cell death (Fig. 9.4).

Collectively, these findings suggest that hair follicle response to moderate laser fluences is characterized by activation of the apoptotic program, a normal physiological process that hair follicle follows during its cycle transition from the anagen to catagen phase. The apoptosis activation is likely to result in premature hair follicle transition to its resting stage, resulting in the cessation of hair growth and achieving a temporary hair removal. The absence of both pro-apoptotic p53 and anti-apoptotic Bcl-2 protein in the hair follicles after exposure to high laser fluences suggests severe and perhaps irreversible damage to the follicular structure, leading to pathological destruction of the hair follicle and permanent reduction in hair.