In Vitro Models for the Evaluation of Anti-acne Technologies

15 May

INTRODUCTION

Acne is the disease manifesting from a multifactorial process  involving the pilose- baceous appendages, and  although the exact mechanism is unknown, four biologi- cal mechanisms are believed to play  critical  roles in the formation of a lesion:

1.    increased sebum production,

2.    aberrant keratinocyte proliferation and  differentiation,

3.    enhanced bacterial growth and  metabolism, and

4.    irritation and  inflammation.

Acne  is unique to humans, and  although animals can be induced to form  come- dones, there is no representative in vivo model  aside from humans, which  incorpor- ates the above-mentioned factors.  Furthermore, of the four  types  of pilosebaceous appendages  described by  Kligman (1),  only  the  sebaceous pilosebaceous unit, which  contains a large sebaceous gland and  a small vellus  hair, has the propensity to develop acne. Even so, acne is still a remarkably rare event, with only a small per- centage of follicles being  diseased at any single  time.

A  detailed  structural  analysis  of  the  follicular  plug,   or  comedone, that can develop into an acne lesion reveals a compacted agglomeration of desquamated follicular  keratinocytes, bacteria, sebaceous gland products/remnants, and hair fibers. Finally,  the static  condition of the comedone allows  for a favorable growth environ- ment  for bacteria, such  as Proprionibacterium acnes and  Staphylococcus epidermidis that can lead to inflammation.

Although no in vivo or in vitro model  exists to examine the entire  complexity of lesion  formation, several  in vitro  models are available to evaluate technologies targeting specific  biological  mechanisms occurring in acne.  This review  is aimed at disclosing the  types  of in vitro  models available to evaluate technologies that may  alleviate or prevent acne lesion  formation.

EVALUATION OF TECHNOLOGIES TARGETING THE SEBACEOUS GLAND

The sebaceous gland of the skin is the part of the pilosebaceous unit, which  secretes a  unique lipid   mixture of  triglycerides,  wax  esters,   and   squalene during  pro- grammed differentiation (2).

The sebaceous gland  consists  solely  of one epithelial cell type,  the sebocyte. Sebocyte  migration out  of the gland’s  basal  layer  and  full differentiation requires approximately two  weeks,  during which  time  the cells increase in volume 100- to

150-fold  and   become   lipid-filled  (3 – 6).  Lipid   secretion into  the  follicular   duct

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then  occurs  via a holocrine mechanism, and  hence  destroying the sebocyte.  There- fore, the absolute rate and amount of sebum secretion is dependent upon sebaceous gland density and size, metabolic activity of the sebocytes, and lipogenic capacity of the ductal reservoir.

The movement of sebum through the follicular  canal and  onto the epidermal surface  allows for interaction with bacterial and epidermal lipases  that can degrade the triglyceride fraction  partially or completely to free fatty acids  (7 – 9).

Most of the literature documenting sebaceous gland physiology has resulted from the use of human volunteer subjects or animal models. Several in vitro models have  been  developed for  further understanding sebocyte  physiology, which  are based  on two  technologies: the in situ  culture of the human sebaceous gland and the  in  vitro  culture of  sebocytes  in  a  monolayer with   and   without  fibroblast feeder  support.

Sebaceous Gland Organ Culture Maintenance

Culture of the  entire  sebaceous gland  offers  several  advantages in comparison to sebocyte  monolayer culture. For example, as the three-dimensional architecture is maintained, proliferation and  lipid  synthesis can be simultaneously examined in the  basal  and   differentiated  cell  layers,   respectively.  An  additional advantage over  animal models lies in the  fact that  delivery of potential sebum suppressive compounds to  the  gland  is not  hindered, so  absolute biological  efficacy  can  be evaluated.

Sebaceous glands can be isolated by dissection of human skin (surgical waste, donor, cadaver, etc.) by a variety of methods including microdissection (10) and shearing (11). Once  obtained, the  glands are  placed directly in  growth medium (or can be floated  by placement upon nitrocellulose filters) and  incubated at 378C in the presence of carbon  dioxide (12). Studies  using  intact  sebaceous glands with and  without the  attached dermal/epidermal components have  been  utilized to measure lipogenesis rates  in  subjects  with  acne  (13) and  the  effect  of substrates on lipid  rates  and  patterns (14 – 16). Acetate  was  determined to be the  most  pre- ferred   substrate resulting in  maximal incorporation  into  newly   formed lipids. Pappas et al. (17) reported that  the  sebaceous gland selectively utilizes the  16:0 fatty  acid  for  incorporation into  wax  esters  and  elongated fatty  acids  such  as

18:0. As a control,  acetate  was  incorporated into  all of the  cellular  and  secreted lipids.

During prolonged incubation at the air/liquid interface, newly  formed sebo- cytes tend  to differentiate similarly as interfollicular keratinocytes taking  on a flat- tened phenotype with  minimal lipid   synthesis occurring. Through  a  thorough investigation of factors  impeding differentiation, Guy  et al. (18,19) reported that many  of the  common growth medium components, such  as phenol red,  HEPES buffer,  epidermal growth factor (EGF), and  serum, reduced overall  lipogenesis. In the  absence  of phenol red  (which  is known to have  estrogen-like properties) and EGF, the intact sebaceous gland can be maintained for up to two weeks without sig- nificant  necrosis  (Fig. 1). DNA,  protein synthesis, and  lipogenesis rates  remained consistent over  the maintenance period.

In the presence of estradiol (600 pM) and  13-cis-retinoic acid (1 mM), lipogen- esis rates  were  decreased on average by 36% and  48%, respectively (Fig. 1). Histo- logically,  a thickening of the  undifferentiated cell layer  accompanied by luminal keratinization was  evident.

FIGURE 1    Organ  culture of the human  sebaceous gland. Sebaceous glands can be maintained in culture over seven days with no discernable loss in proliferation or lipid synthesis. (A) Freshly isolated human  sebaceous gland.  (B) Sebaceous gland maintained in culture for seven days.  (C) As in (B), but  supplemented with 600 pM 17-b-estradiol. Rates  of cell  division  are  comparable to  control but lipid synthesis is decreased on average by 41%.  (D) As in  (B), but supplemented with 1 mM

13-cis-retinoic acid.  Rates of cell  division  and  lipid synthesis are  decreased by  36%  and  48%,

respectively. Source: From Ref. 19.

Surprisingly, testosterone and  dihydrotestosterone (DHT)  had  no  effect  on enhancing lipogenesis in  the  cultured glands, possibly because the  glands were already at maximal lipogenesis rates.  This finding implied that  androgens are not necessary for  the  short-term stimulation of glandular function or,  alternatively, that   the  growth  conditions for  sebaceous gland   maintenance  have   been  fully optimized.

Additionally, peroxisome proliferator activated receptors (PPARs) have  been shown to be important in the regulation of lipid  metabolism in adipose and  liver cells.  PPARs  are  nuclear hormone receptors and  sebocytes express the  subtypes a, b, and  g that  appear to play  important roles in proliferation and  differentiation. Downie et al. (20,21) investigated the role of PPAR and additional nuclear hormone receptor ligands on sebaceous gland proliferation and  differentiation.

PPARa,  PPARb,   PPARg,  retinoid-X-activated receptor  a,  liver-X-activated receptor  a,  and   pregnane-X-receptor,  but   not   farnesoid-X-activated  receptors, were  all detected in  fresh  and  seven-day maintained glands. The  PPAR  ligands arachidonic  acid,  BRL-49653, clofibrate,   prostaglandin J2, eicosatetraynoic acid (EYTA), leukotriene B4, linoleic  acid,  linolenic  acid,  oleic acid,  and  WY-14643 all inhibited lipid  synthesis, whereas bezafibrate, juvenile  hormone III, and  farnesol were ineffective. With the exception of EYTA at 10 mM, no effects on gland  prolifer- ation  were  evident (20,21).

The sebaceous gland in situ model  is also applicable for the culture of sebac- eous  glands isolated from  animals. Toyoda  and  Morohashi (22) performed organ

culture on sebaceous glands obtained from mice to evaluate the effects of neuropep- tides  (calcitonin gene-related peptide, substance P, vasoactive intestinal polypep- tide,  and  neuropeptide Y) in  addition to nerve  growth factor.  Sebaceous glands treated with  substance P resulted in accelerated lipid  synthesis over  the  control glands by increasing the  rate  of proliferation and  differentiation postulating that stress  could  participate in the pathogenesis of acne.

Sebocyte Monolayer Culture

Although the  advantages of sebaceous gland organ  culture are  evident, the  pro- cedure is time-consuming and  dependent on  continued sources  of viable  tissue for additional glands. Cell culture offers an alternative over organ  culture mainten- ance, as large numbers of cells can be processed and  frozen  back allowing multiple experiments to be performed on a similar cell lineage.  However, continued holo- crine  destruction of sebocytes also  necessitates a  stem  cell  population required for maintenance. Stem cells are self-renewing and  are capable of generating large numbers  of  differentiated progeny  throughout  an  individual’s  life  span   (23). Stem cells can also generate a transit amplifying population, which  has a high prob- ability  of postmitotic differentiation. Within  the  human sebaceous gland,  transit amplifying cells  have  been  detected (24,25). For  a recent  review  on  hair  follicle stem  cells, refer to Lavker  et al. (26).

The  in  vivo  data  appeared consistent with  early  in  vitro  experiments that failed  to yield  viable  sebocytes during extended culture conditions (27 – 29). This resulted in a similar but  less  stringent need  as organ  culture for ample biopsies to supply sufficient  sebocytes for experimentation.

Several  laboratories have  successfully cultured human sebocytes in the pre- sence  of fibroblast support and  in serum-free conditions to examine the effects of serum, growth factors,  and  biopsy  location  (30 – 33). Sebocyte  culture provides an excellent  model to change  local environmental growth conditions (i.e., hormones, vitamins, carbohydrates, etc.)  and  to  evaluate lipogenesis inhibitors in  a higher throughput mode  than  that  which  could  normally be obtained through the use of organ   culture maintenance. This  can  be  accomplished either  through extended culture  to  evaluate  gross   morphological  changes  (i.e.,  sebocyte   size,   keratin expression patterns, and  lipid  vacuole content) or through rapid evaluation using radiolabeled substrates. Tritiated thymidine will be incorporated into  the  cells as a marker for  proliferation, whereas radiolabeled glucose  and/or  acetate  can  be used  as tracers  for lipid  synthesis.

Two procedures are suitable for generating sebocyte  monolayer cultures. In the  explant outgrowth method, isolated sebaceous glands are attached to culture plates   by  limited drying  followed by  addition of  growth  medium.  Fibroblast- feeder  support can  later  be  added, but  this  appears not  to  be  necessary. After several  days  of incubation, proliferative basal  sebocytes are  seen  as  visible  out- growths from the gland  (Fig. 2).

Alternatively, a much  more  common approach is to digest  sebaceous glands by limited trypsin proteolysis and  plate  the released cells in mass  culture upon a fibroblast feeder  layer.  After  several  days,  colonies  of attached cells will begin  to proliferate into small  colonies  that  can be further cloned  and/or passed.

During the proliferative stage, sebocytes are indistinguishable from keratino- cytes  expressing keratins 5 and  14. Upon  reaching confluence, the  cells take  on a sebocytic  phenotype as  they  enlarge, express keratins 4, 7, and  13, and  become

FIGURE 2    Primary  culture  of human  sebocytes derived  from explant  growth.  Human  sebocytes can  be propagated in monolayer culture by outgrowth  of sebaceous glands through  in vitro explant culture.  (A, inset) Colony size  at day 5. (B, main photograph) Colony size  at day 10. Source: From Ref. 31.

lipid filled. Detection of an intracellular lipid droplet is evident by retention of lipo- philic  dyes  such  as Nile red,  oil red  O, or Sudan  black (Fig. 3). Sebocytes,  in com- parison to cultured keratinocytes, synthesize and  accumulate considerably more intracellular lipids  at comparable stages  of differentiation (Fig. 4), and  analysis by thin  layer  chromatography (TLC) reveals  similar classes  of sebum-specific lipids as those  made de novo  (Fig. 5).

Although sebocytes can  be cultured for an  extensive passage number (34), there   may   be  a  tendency  for   sebaceous  cells   to  de-differentiate  to  varying degrees, especially in regard to sebum specific lipids.  The rate-limiting step in squa- lene  synthesis shifts,  as the  cells tend  to accumulate higher levels  of cholesterol, which  is then  esterified into  cholesterol esters.  This  de-differentiation appears to have  been  corrected by  Zouboulis et  al.  (35), when freshly   isolated cells  were immortalized  by  transfection  with   a  PBR-322  based   plasmid  containing the coding  region of the simian  virus-40 large T antigen. These sebocytes have been pas- saged  over 50 times  with  no discernable loss of phenotype (Fig. 6).

Regardless of the culture conditions being utilized, sebocyte  monolayer culture has been used successfully to answer many  questions regarding cell metabolism and lipid  synthesis, specifically the androgen metabolism and  retinoid pathways.

Fujie et al. (31) reported that the androgens testosterone and  DHT stimulated the  proliferation as well  as the  lipid  synthesis of sebocytes in a dose-dependent manner with  maximal effects occurring at 1029  and  10210  M, respectively. Zoubou- lis et al. (32) found that testosterone stimulated facial sebocyte proliferation by 50% at 1026  M and  DHT at 1028  M. In the presence of the antiandrogen spironolactone, sebocyte  proliferation was  reduced by 50% at 1027 M and  additionally, the stimu- latory  effect seen with  DHT was neutralized. Spironolactone has been successfully used  as a treatment for acne (36,37), suggesting that  the inhibitory effects of anti- androgens may  be occurring at the cellular  level.

The stimulatory effect seen with  androgens in sebocyte  monolayer culture is in contrast to the results seen in organ culture in which  no effects were evident. This

FIGURE 3    The in vitro culture of human sebocytes. Human sebocytes can be cultured in vitro under a variety of conditions  including the presence/absence of serum, phenol  red, and fibroblast support to attain sebocyte-specific differentiation characteristics. (A) Preconfluent human  sebocytes cultured in vitro in serum-free medium  in the absence of fibroblast support. (B) Sebocytes cultured  in phenol red-free  and   serum-free  medium   at   seven  days   postconfluence.  Note   the   accumulation  of intracellular  lipid droplets. (C)  Sebocytes at  seven days  postconfluence stained with oil red  O confirming the presence of intracellular  lipids.

may be due  to a dilution of inherent androgens, as sebocytes multiply to a greater extent in monolayer culture. However, it has been documented that sebocytes are able to  synthesize testosterone from  adrenal precursors in  parallel to  an  inactivation process  to maintain androgen homeostasis (38). Alternatively, the presence of ingre- dients in the growth medium such  as phenol red,  which  has inhibitory activity  via estrogen-like properties, may account for the stimulatory effects seen with androgens. It is suggested that  all  experiments with  cultured sebocytes be  performed using phenol red-free medium under reduced lighting conditions for optimal benefits.

FIGURE 4    Oil red  O staining  of sebocyte and  keratinocyte cultures. Upon  reaching confluence, sebocyte differentiation  involves  synthesis and  accumulation of intracellular  lipid droplets. (Left): Human  sebocytes cultured  in vitro for seven days  postconfluence in serum-free/phenol red-free medium   (60 mm2  area).  Note  the  extensive oil red  O  staining   within  the  dish  confirming  the presence of  lipids.  (Right):  Similar  aged culture   of  human   keratinocytes stained  with  oil red O. Note the absence of significant intracellular  lipids.

It is well-known that  oral administration of 13-cis-retinoic  acid reduces sebaceous gland size  and  lipid  synthesis, whereas the  all-trans  and  9-cis isomers of retinoic  acid  are  significantly less effective.  Therefore, cultured sebocytes have also  been  utilized as a model  to further understand retinoid responsiveness and potential mechanism of action.

Tsukada et al. (39) reported that  isomerization of 13-cis-retinoic  acid  to all- trans retinoic  acid occurs in SZ-95 sebocytes, subsequently providing the biological benefit.  No  isomerization was  evident in  HaCaT  keratinocytes and  the  reverse

FIGURE 6    Culture  of immortalized  sebocytes. Human  facial  sebocytes can  be  immortalized  to retain  sebocyte-specific differentiation  characteristics by transfection with  simian   virus-40   large T antigen. The  resulting  cell line (SZ-95)  was  further  cloned  and  extensively passaged  with no discernible loss  of features. (A) Preconfluent cultures of human  sebocytes prior to transfection. (B)  First  passage of SZ-95  immortalized  sebocytes. (C)  SZ-95  immortalized  sebocytes at  50th passage. Note the appearance similar to sebocytes from first passage. Source: From Ref. 35.

conversion of all-trans  to  13-cis-retinoic  acid  was  not  evident in  sebocytes. Both

13-cis and   all-trans   retinoic   acid  suppressed mRNA   expression of  cytochrome P450-1A2, and  in  addition, 13-cis, all-trans,  and  9-cis-retinoic  acid  reduced sebo- cyte  proliferation on average by 40% at 1027 M. Retinoids also  do  not  appear to effect the programmed cell death of human sebocytes (40).

In additional studies, Zouboulis et al. (41,42), using  nontransfected sebocytes, reported that  13-cis-retinoic  acid  reduced radiolabeled acetate   incorporation  by

48%, whereas all-trans  and  acitretin only  reduced incorporation by 38% and  27%, respectively.

Additional reports using  immortalized sebocytes have also been documented to measure expression of steroidal enzymes (43), PPARs, and transcriptional factors (44), in addition to melanocortin receptors and the effects of select interleukins (ILs) (45). Finally,  the patent databases are an excellent  source  of reviewing intellectual property on sebum suppressive technologies.

Rat  Preputial Sebocyte Monolayer Culture

The rodent preputial gland  has also been used  as a model  for the human sebaceous gland.  These  specialized glands open  to the surface  on either  side  of the urethral meatus, and secretions are involved in territorial marking and mating behavior (46).

Using techniques similar to that used with human sebaceous glands, rat prepu- tial glands can be isolated, digested, and  cultured upon a fibroblast feeder  layer  to produce monolayer cultures (47,48) that can be manipulated and studied. Rosenfield (49) reported on the relationship of sebaceous cell stage with growth in culture in that

fully  differentiated  sebocytes are  not  capable of  attachment and   proliferation, whereas early differentiated cells are. Subsequent studies by Laurent et al. (48) docu- mented the requirement of serum and  growth factors  to enhance growth and  that preputial  sebocytes also   expressed  cytokeratin-4 and   formed  fewer   cornified envelopes than  keratinocytes. The  effects  of estrogen on  preputial cell behavior (50) and  characterization of the cultured cells (51,52) along  with  the mechanism of androgen action (53) and  effects of PPARs (54) have also been reported.

Preputial cells differentiate in a similar process, but to an overall  lesser extent than   human  sebocytes even  in  the  presence of  androgens (Fig.  7).  Rosenfield et  al.  (55) discovered that  preputial cells  cultured in  vitro  lack  the  presence  of PPAR   ligands  that   will   induce  lipid   droplet  formation. The  effect  of  PPAR ligands was  found to be distinct and  additive to the  effect  seen  with  androgens in increasing lipid  droplet formation.

FIGURE 7    Differentiation   of  preputial   sebocytes.  Preputial   sebocytes  can   be   cultured   in vitro   with   3T3-fibroblast   support.  Differentiated    sebocytes  containing    lipids   are   positively identified  via  staining  with  oil  red   O.  (Top  left):  Oil  red   O  staining  of  preputial  sebocytes cultured  in vitro for nine  days  in which  11%  of the  culture has  differentiated into lipid-forming colonies.  (Lower  left):  Oil  red  O  staining  of  preputial   sebocytes cultured   in  the  presence of

1026  M dihydrotestosterone  (DHT)  in  which  25%  of  the  culture  has   differentiated  into  lipid-

forming   colonies.  (Top   right):  Oil  red   O   staining  of  preputial  sebocytes  cultured  in  the presence of  1026  M peroxisome  proliferator  activated  receptor  g  ligand  BRL-49653  in  which

66%   of  the   culture   has   differentiated  into  lipid-forming  colonies.  (Lower  right):  Oil  red   O staining   of  preputial   sebocytes  cultured   in  the  presence of  1026 M DHT  and  BRL-49653   in which 80% of the culture has  differentiated into lipid-forming colonies. Abbreviation: DHT, dihydrotestosterone. Source: From  Ref.  53.

Kim et al. (56), furthermore, investigated the  effect of retinoids on prepu- tial  cell  growth  and   differentiation.  All-trans   retinoic   acid   and   retinoic   acid receptor  agonists  resulted  in   a  decrease  in   cell  number,  colony   size,   and number of  lipid-forming colonies.  Kim  concluded that  retinoic   acid  receptors and  retinoid-X-receptors play  select  roles  in sebocyte  proliferation and differentiation.

Other In Vitro Sebaceous-Cell-Based  Models

Animal models such  as the hamster flank organ  and  hamster ear have  been devel- oped  to study the effects of anti-acne and  sebum-suppressive capabilities of com- pounds and   formulations  in  vivo.  Following application,  the  target   areas   are excised,   weighed, and  processed for  histological examination.  In  other  animal models, monitoring of sebum secretions follows intravenous or oral administration of sebum-suppressive agents.

The  hamster auricle  sebaceous gland has  been  successfully cultured  and utilized  as  a  model   for  human  sebaceous glands.  Glands  are   isolated  and cells  are  grown on  a  3T3 fibroblast-feeder layer  using  standard  protocols. Ito et  al.  (57),  Sato  et  al.  (58),  and   Akimoto  et  al.  (59)  have   reported  that   the amount of lipid  produced increases following the peak  of sebocyte  proliferation and  that  androgens stimulate, whereas retinoic  acid,  EGF, and  vitamin D3  sup- press  overall  activity.

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