|Year : 2013 | Volume
| Issue : 2 | Page : 91-97
Immunolocolization of CD29 in keloid
Mohamed A Basha1, Rehab M Samaka2, Ola Bakry1, Iman M Elwan1
1 Department of Dermatology, Andrology and Sexually Transmitted Diseases, Shebin Elkom, Egypt
2 Department of Pathology, Faculty of Medicine, Menoufiya University, Shebin Elkom, Egypt
|Date of Submission||21-May-2013|
|Date of Acceptance||21-Jul-2013|
|Date of Web Publication||31-Jan-2014|
Iman M Elwan
MB, BCh, Department of Dermatology, Fuwa Hospital, Fuwa, Kafr El-Sheikh
Source of Support: None, Conflict of Interest: None
To shed light on the role of mesenchymal stem cells (MSCs) in keloid pathogenesis.
Keloids are fibroproliferative scars that show a benign yet quasineoplastic behavior and morphology, as evidenced by high recurrence and aggressive invasion into the surrounding skin. MSC populations originate from bone marrow (BM) stroma, which can give rise to stromal mature mesenchymal cells and aid regeneration of the BM microenvironment. Human BM-derived MSCs may contribute toward keloid pathogenesis.
Patients and methods
A total of 30 skin biopsy specimens from keloid lesions were used and 15 healthy volunteers were used as a control. Histopathological examination of hematoxylin and eosin-stained sections of keloid was carried out for the evaluation of histopathological parameters. The expression of CD29 was examined immunohistochemically.
There were statistically significant differences between CD29 immunostaining in normal skin and lesional area of keloid in favor of keloid. There were no significant differences between different epidermal intensities of CD29 expression in keloid.
MSCs may share in keloid pathogenesis. The future therapy of keloid scars may have to target MSC differentially in order to deprive these tumors of their regenerative cell pools. This may represent an innovative method for keloid treatment.
Keywords: CD29, keloid, mesenchymal stem cells
|How to cite this article:|
Basha MA, Samaka RM, Bakry O, Elwan IM. Immunolocolization of CD29 in keloid. Menoufia Med J 2013;26:91-7
| Introduction|| |
Keloids are fibroproliferative scars that show a benign yet quasineoplastic behavior and morphology, as evidenced by high recurrence and aggressive invasion into the surrounding skin  . Dark-skinned individuals develop keloids 15 times more frequently than do their lighter-skinned counterparts  . Although keloid can occur at any age, they are most likely to occur between the ages of 11 and 30 years  . A slight female predominance is also noted, but this could be related to the higher rate of ear lobe piercing in females  .
Research continues to identify the genetic, cellular, immunological, and biochemical pathways, in addition to environmental factors, responsible for the development of keloids  .
Keloid tissue has been shown to be more metabolically active and to use more oxygen than normal scar tissue. This leads to a relative state of hypoxia in keloid fibroblasts. This high oxygen-consuming potential and low oxygen diffusion may contribute toward the pathophysiology of keloid formation  .
Stem cells are unspecialized cells that have two defining properties: the ability to differentiate into other cells and the ability to self-regenerate  .
Bone marrow (BM)-derived cells can be divided into hematopoietic stem cells and mesenchymal stem cells (MSCs)  . MSC populations from BM stroma, which can give rise to stromal mature mesenchymal cells and regenerate the BM microenvironment, have been identified in a perivascular region in BM tissue  .
Adult stem cells may play an essential role in growth, homeostasis, and regeneration of many tissues. However, to date, how stem cells contribute toward pathologic diseases remains largely unknown. Several studies have reported the isolation and characterization of precursor cells from normal rodent and human normal skin dermis (SKPs)  and, recently, mesenchymal-like stem cells from keloid scar (KMLSCs)  . These so-named KMLSCs also shared the most important characteristic of MSCs, namely, the capacity to differentiate into mesenchymal lineage (osteogenic, chondrogenic, and adipogenic) cells. In addition, KMLSCs could differentiate into endothelial cells and smooth muscle-like phenotypes in vitro, which has also been observed for BM-derived and placenta-derived MSCs  .
CD29 is an integrin unit associated with very late antigen receptors. It is MSC marker. It is known to conjoin with the α3 subunit to create the α3μ1 complex, which reacts with such molecules as netrin-1 and reelin  . Integrins are noncovalently associated heterodimers of 1α and 1μ subunit, altogether forming more than 24 integrins using 18α and 8μ subunits  . Both the α and μ subunits are type-I membrane proteins with a large extracellular domain and a generally short, noncatalytic cytoplasmic tail, linked by a single transmembrane region  . Integrins are important for cell-cell and extracellular matrix (ECM) interactions  .
| Aim of the work|| |
The aim of this work was to elucidate the role of mesenchymal-like stem cell (MLSC) in the pathogenesis of keloid through immunohistochemical localization of these cells in keloid tissue.
| Patients and methods|| |
This study was carried out on 30 patients with keloid and 15 healthy volunteers as a control. The patients were recruited from the Dermatology and Plastic Surgery Outpatient Clinic, Faculty of Medicine, Menofiya University, and Shibin El Kom Educational Hospital. All patients gave their formal consent. The protocol was approved the Ethical Committe of the Faculty of Medicine, Menofiya University.
The diagnosis of keloid was made on the basis of patients' history and the typical clinical features that were confirmed by histopathological examination. Patients stopped medical treatment, either topical or systemic, 2 weeks before the biopsy.
Each of the selected patients was subjected to the following:
- Complete history taking.
- Clinical examination.
- Skin biopsy: After obtaining written consent from the patients, biopsies were taken under local anesthesia (from the lesion and normal skin). All biopsies were fixed in 10% neutral-buffered formalin, dehydrated in ascending grades of ethanol, followed by xylene, and then impregnated in paraffin. Two 5-μm thick sections from each block were obtained. One section was stained by hematoxylin and eosin for histopathological examination. The other sections were mounted on Superfrost Plus slides (Fisher Scientific) and stored at room temperature until used for immunostaining.
Histological examination of skin from the patients studied showed a wide range of pathological findings. Histological examination of keloid was carried out according to Manchester's histopathological assessment sheet. Keloidal scar showed that epithelial thickness was decreased and rete ridges were lacking. Cellularity and vascularity were increased, but hair follicles and sebaceous glands were lacking. Collagen fibers were disoriented and showed increased density.
Immunohistochemical staining for CD29
For CD29 (Integrin μ-1) (Clone 7F10) immunostaining, Mouse Monoclonal Antibody (Cat. #MS-1089-R7) was used (Thermo Fisher Scientific, Fremont, California, USA). Positive control was tonsil.
The immunostaining technique applied in this study was the improved streptavidin-biotin amplified system.
In this system, three reagents were utilized: the primary antibody specific for the antigen to be localized, the biotinylated secondary anti-immunoglobulin that is capable of binding to both the primary antibody, and the streptavidin-biotin enzyme complex. Finally, the reaction can be visualized by an appropriate substrate/chromogen reagent that is called diaminobenzidine (DAB). Antibody concentration was ready to use and the source was Thermo Clone 7F10.
Interpretation of CD29 expression
Positive immunostaining of CD29 was identified by brownish membranous discoloration of the cell membrane  [Figure 1] and [Figure 2].
Results were collected, tabulated, and analyzed statistically using an IBM personal computer and the statistical package SPSS, version 11. Fisher's exact test and χ2 -test were used to study the association between two qualitative variables. The Mann-Whitney U-test was used for comparison of quantitative variables that were not normally distributed. A P value of 0.05 or less was considered statistically significant.
| Results|| |
There were significant differences between normal skin and lesional skin in CD29 epidermal intensity and the epidermal H score (P < 0.001 for both), whereas the lesional area had higher intensities and H scores than normal skin [Table 1]. Similarly, significant differences were found between lesional skin and normal skin in CD29-positive dermal expression, dermal H score, and dermal intensity (P < 0.001 for all) [Table 1].
|Table 1: Comparison between lesional areas of keloid cases and the control group in terms of CD29 immunostaining|
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There were no significant differences between different epidermal intensities of CD29 expression in keloid cases in the demographic, clinical, and histopathological characteristics [Table 2]. There were no significant differences between different dermal intensities of CD29 expression in the demographic, clinical, and histopathological characteristics of the studied cases [Table 2].
|Table 2: Relationship between different epidermal and dermal intensities of CD29 expression and the demographic, clinical, and histopathological characteristics of keloid cases|
Click here to view
| Discussion|| |
Keloids show a benign yet quasineoplastic behavior and morphology  , as evidenced by high recurrence and aggressive invasion into the surrounding skin and because they show cancer-like bioenergetics of keloid fibroblasts in vitro  . Given that the contribution of intracutaneous adult stem cells, namely of resident, so-called MLSCs, toward skin tumor formation is increasingly being discussed  , it appears rational to investigate the potential role of MSCs in keloid pathogenesis  .
It was suggested that the interaction between stem cells and keloid-derived fibroblasts may contribute to keloid formation by triggering progressive myofibroblastic differentiation and secretion of abundant ECM  .
The current work showed an upregulation of CD29 in lesional keloid tissue relative to normal skin. Our findings were in agreement with previous studies that have shown the presence of MLSCs in keloid disease , .
CD29 is normally expressed on keratinocytes, endothelial cells, fibroblasts, and T lymphocytes. It acts as a receptor for extracellular matrix components and is important for the attachment of cells to connective tissue constituents and may act to facilitate the migration of lymphocytes and other inflammatory cells in normal and diseased cutaneous conditions  .
Iqbal et al.  reported an upregulation of CD29 in lesional keloid tissue relative to normal skin. The authors postulated that keloid scars themselves seem to provide a niche environment for nonhematopoietic MSC. Further studies are recommended for a more conclusive outcome.
Human BM-derived MSCs (hMSCs) may contribute toward keloid pathogenesis as shown by rich collagen production in their cytoplasms  . This occurs through the production of extracellular matrices and, thus, causes the extension and augmentation of keloid formation. These phenomena may be implicated in the recurrence of keloids in the same area or the exacerbation of preexisting keloids through the blood stream  .
The origin of MSCs in keloid tissue has received considerable debate. Akino et al.  reported that keloid-derived fibroblasts could induce MSC chemoattraction toward keloid cells, whereas normal fibroblast failed to be chemoattracted to keloid-derived fibroblasts. In addition, keloid fibroblasts or keloid-derived humoral factors induced MSC differentiation  .
Keloid is characterized by chronic inflammation, an increased infiltration of inflammatory cells, an enriched milieu of cytokines and growth factors, and an abundant accumulation of ECM  , thus providing a unique inflammatory niche that generates homing signals to recruit BMMSCs to sites of injury and inflammation  , whereby a variety of inflammatory mediators, including hypoxia, reactive oxygen species, inflammatory chemokines, and cytokines, may trigger the migration of BMMSCs  . In response to the unique tumor microenvironment of chronic inflammation  , BMMSCs may acquire the cancer-associated fibroblast phenotype and subsequently promote tumor growth and metastasis through the production of proangiogenic and tumor-stimulating paracrine factors  .
These findings suggest that the unique keloid microenvironment or niche may preferentially recruit BMMSCs and sustain their interaction with resident fibroblasts, thus contributing toward keloid formation by producing abundant extracellular matrix at the scar site  .
Supporting the role of inflammatory niche in keloid pathogenesis, it was postulated that the fibroblasts in the keloid dermis may actually be multipotent stem cells that are maintained in a proliferating and undifferentiated state by a particular local cytokine milieu and they can be maintained in a multipotent and proliferative state by aberrant local conditions  .
Supporting this notion, it has been shown that keloid fibroblasts proliferate abnormally  and that some keloid fibroblast characteristics are lost when these cells are removed from the in-vivo microenvironment of the keloid lesion and passaged in vitro  .
The increased fibroblast proliferative activity in the keloid may normalize if the cytokine milieu surrounding the keloid is manipulated appropriately  . When these cells were isolated, they showed a typical fibroblast-like morphology, growth rate, and cell cycle regulatory gene and adhesion molecule expression profile; they expressed classical MSC marker proteins (but not hematopoietic and endothelial markers)  .
It was proved that the dermal layer harbors embryonic and MSCs that are randomly distributed in the reticular dermis and interspersed among thick ECM. Therefore, the presence of postnatal dermal stem cells may share in pathologic scar and may contribute toward its persistent growth  .
In the last few decades, several groups have shown that multipotent precursor cells can be generated from different normal adult human tissues  and may share common biological properties, including the expression profile of various cell surface markers and embryonic transcription markers as well as their multipotent differentiation capabilities  .
Thus, adult stem cells may play an essential role in the growth, homeostasis, and regeneration of many tissues. However, to date, how stem cells contribute toward pathologic diseases remains largely unknown. Several studies have reported the isolation and characterization of precursor cells from normal rodent and human normal skin dermis (SKPs)  and recently, KMLSCs  .
Such skin-derived precursors (SKPs) appear to be multipotent because SKPs isolated and expanded from rodent and human skin can differentiate into mesenchymal lineage cells as well as neural progeny  . Thus, these cells may be useful for replacing not just skin tissue but tissues in other organs as well  .
These so-named KMLSCs also shared the most important characteristic of MSCs, namely, the capacity to differentiate into mesenchymal lineage (osteogenic, chondrogenic, and adipogenic) cells. In addition, KMLSCs could differentiate into endothelial cells and smooth muscle-like phenotypes in vitro, which has also been observed for BM-derived and placenta-derived MSCs  .
Fernandes et al.  concluded that KMLSCs also closely resemble SKPs in other ways. First, undifferentiated KMLSCs, similar to SKPs, express embryonic transcription factors that are characteristic of embryonic neural crest precursor cells. Second, KMLSC spheres are very similar to SKP spheres. These observations together suggest that, KMLSCs, similar to SKPs, are derived from neural crest-like precursor cells that appear in the skin during late embryogenesis, probably through migration, after which they persist in lower numbers in adult skin  .
It was reported previously that KMLSCs may represent an active, benign, tumor-like stem cell counterpart of SKPs that might contribute, at least in part, toward the high proliferative state in keloid tumor  .
| Conclusion|| |
MSCs may share in keloid pathogenesis. The future therapy of keloid scars may have to target differentially MSC in order to deprive these tumors of their regenerative cell pools. This may represent an innovative method for keloid treatment.
| Acknowledgements|| |
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2]