Barbara Shih

Molecular dissection of abnormal wound healing processes resulting in keloid disease

Keloids are locally aggressive scars that typically invade into healthy surrounding skin and cause both physical and psychosocial distress to the patient. These pathological scars occur following minimal skin trauma after a variety of causes including burns and trauma. Although the pathogenesis of keloid disease is not well understood, it is considered to be the end product of an abnormal healing process. The aim of this review was to investigate the molecular and cellular pathobiology of keloid disease in relation to the normal wound healing process. The molecular aberrances in keloids that correlate with the molecular mechanisms in normal wound healing can be categorized into three groups: (1) extracellular matrix proteins and their degradation, (2) cytokines and growth factors, and (3) apoptotic pathways. With respect to cellular involvements, fibroblasts are the most well‐studied cell population. However, it is unclear whether the fibroblast is the causative cell; they are modulated by other cell populations in wound repair, such as keratinocytes and macrophages. This review presents a detailed account of individual phases of the healing process and how they may potentially be implicated in aberrant raised scar formation, which may help in clarifying the mechanisms involved in keloid disease pathogenesis.

Genetics of keloid scarring.

Keloid scarring, also known as keloid disease (KD), is a common, abnormally raised fibroproliferative cutaneous lesion that can occur following even minor skin trauma. The aetiopathogenesis of KD has remained an enigma todate compounded by an ill-defined clinical management. There is strong evidence suggesting a genetic susceptibility in individuals affected by KD, including familial heritability, common occurrence in twins and high prevalence in certain ethnic populations. This review aims to address the genetic aspects of KD that have been described in present literature that include inheritance patterns, linkage studies, case–control association studies, whole genome gene expression microarray studies and gene pathways that were significant in KD. In addition to our clinical and scientific background in KD, we used search engines, Scopus, Scirus and PubMed, which searched for key terms covering various genetic aspects of KD. Additionally, genes reported in seven whole genome gene expression microarray studies were separately compared in detail. Our findings indicate a varied inheritance pattern in KD (predominantly autosomal dominant), linkage loci (chromosomes 2q23 and 7p11), several human leukocyte antigen (HLA) alleles (HLA-DRB1*15, HLA-DQA1*0104, DQ-B1*0501 and DQB1*0503), negative candidate gene case–control association studies and at least 25 dysregulated genes reported in multiple microarray studies. The major pathways reportedly proposed to be involved in KD include apoptosis, mitogen-activated protein kinase, transforming growth factor-β, interleukin-6 and plasminogen activator inhibitor-1. In summary, involvement of more than one gene is likely to be responsible for susceptibility to KD. A better understanding of the genes involved in KD may potentially lead to the development of more effective diagnostic, therapeutic and prognostic measures.

Scientific understanding and clinical management of Dupuytren disease

Dupuytren disease (DD) is a fibroproliferative disorder of unknown etiology that often results in shortening and thickening of the palmar fascia, leading to permanent and irreversible flexion contracture of the digits. This Review provides a detailed update of the scientific understanding of DD and its clinical management, with perspectives on emerging research and therapy. Established risk factors include genetic predisposition and ethnicity, as well as sex and age. Several environmental risk factors (some considered controversial) include smoking, alcohol intake, trauma, diabetes, epilepsy and use of anticonvulsant drugs, and exposure to vibration. DD has been variously attributed to the presence of oxygen free radicals, trauma to the palmar fascia, or aberrant immune responses with altered antigen presentation, or to interactions between these proposed mechanisms. The presence of immune cells and related phenomena in DD-affected tissue suggests that DD is possibly immune-related. Mechanically, digital contracture is caused by myofibroblasts in the DD palmar fascia; however, the exact origin of this cell type remains unknown. The mainstay of treatment is surgical release or excision of the affected palmodigital tissue, but symptoms often recur. Nonsurgical correction of DD contractures can be achieved by Clostridium histolyticum collagenase injection, although the long-term safety and recurrence rate of this procedure requires further assessment.

Identification of Biomarkers in Dupuytren's Disease by Comparative Analysis of Fibroblasts Versus Tissue Biopsies in Disease-Specific Phenotypes

PURPOSE Biomarkers are molecular mediators that can serve as indicators of normal biological processes, pathologic processes, and therapeutic interventions. This study aims to identify potential biomarkers in Dupuytrens disease (DD), a fibroproliferative benign tumor with an unknown etiology and high recurrence after surgery. METHODS Bioinformatic analytical techniques were employed to identify candidate genes that may be differentially expressed in DD, which included gene expression analysis of microarray data and thorough literature searches in genetic linkage and other related biomolecular studies. All DD cases were males with advanced DD (n = 5, 66 years +/- 14). RNA was extracted from biopsies and corresponding cultures of normal fascia (unaffected transverse palmar fascia), palmar nodule and cord from each patient. Real-time reverse transcription-polymerase chain reactions were performed to determine the gene expression levels for disease-related transcripts. RESULTS The bioinformatic analysis revealed 25 candidate genes, which were further short-listed to 6 genes via functional annotation. The 6 selected candidate genes included: A disintegrin and metalloproteinase domain (ADAM12), aldehyde dehydrogenase 1 family member (ALDH1) A1, Iroquois homeobox protein 6 (IRX6), proteoglycan 4 (PRG4), tenascin C (TNC), and periostin (POSTN). The culturing treatments were shown to have significant impact on the gene expression for ALDH1A1, PRG4, and TNC. In tissue biopsies, significant fold changes were observed for ADAM12, POSTN, and TNC in the cord and/or nodule when compared with that of normal fascia. ADAM12 and POSTN are associated with accelerated or abnormal cell growth, whereas TNC has been associated with fibrotic diseases and cell migration. CONCLUSIONS This study demonstrated differential gene expression results in DD tissue biopsies compared with that of their corresponding cultures. ADAM12, POSTN, and TNC were identified from the cord and nodule biopsy samples as potential biomarkers in relation to DD development.

Differential gene expression analysis of subcutaneous fat, fascia, and skin overlying a Dupuytren's disease nodule in comparison to control tissue.

Dupuytren’s disease (DD) is a benign fibroproliferative tumor with an unknown etiology and high recurrence postsurgery. Several observations suggest the possible involvement of skin overlying nodule (SON) and the subcutaneous fat in the pathogenesis of DD. This study aims to (1) compare the gene expression levels of SON and subcutaneous fat in DD and normal subjects and (2) to compare transverse palmar fascia (Skoog’s fibers) from DD patients as internal control tissue, with palmar fascia (transverse carpal ligament) from patients undergoing carpal tunnel release as external control. Skin, fat, and fascia were obtained from five DD patients of Caucasian origin (age = 66 ± 14) and from five control subjects (age = 57 ± 19) undergoing carpal tunnel release. Total ribonucleic acids was extracted from each sample and used for complementary deoxyribonucleic acid synthesis. Real-time quantitative polymerase chain reaction was used to assess the gene expression levels of six candidate genes: A disintegrin and metalloproteinase domain (ADAM12), aldehyde dehydrogenase 1 family member A1 (ALDH1A1), iroquois homeoboxprotein 6 (IRX6), periostin, osteoblast specific factor, proteoglycan 4, and tenascin C. Using independent t test, ADAM12, ALDH1A1, and IRX6 expression levels in DD fats were significantly (p < 0.05) higher than those in the controls. There is no significant difference in the gene expression levels of all six genes when comparing disease and control fascia and skin. Interestingly, ADAM12 up-regulation has also been observed in several other fibrotic and proliferative disorders. In conclusion, this study demonstrates potential roles for subcutaneous fat in DD pathogenesis as well as supports the use of transverse palmar fascia as appropriate control tissues in DD research.

Whole genome and global expression profiling of Dupuytren's disease: systematic review of current findings and future perspectives

Dupuytrens disease (DD) is a common fibroproliferative disorder affecting the palmar fascia, which may lead to permanent contracture of the affected digit. Profiling studies investigating DD at whole-genomic, transcriptomic and proteomic levels have been carried out, from which large numbers of candidate genes potentially involved in DD have been reported. This review focuses on identifying genes reported by multiple studies or validated by multiple experimental techniques, as well as signalling pathways suggested to contribute to DD. Meta-analysis was also carried out on three microarray datasets. Twenty-one genes were found to be reported as dysregulated in multiple gene expression microarrays, seven of which have been further validated by other experimental methods. Sixty-four genes determined to be dsyregulated by meta-analysis correlate to those reported by published microarray studies. In addition, several pathways have been proposed to be involved in DD by whole-genome or global expression profiling. Further investigation in these genes and pathways, and correlating them to genotypes or environmental factors for DD, may aid in further elucidation of mechanisms involved in DD pathogenesis.

Identification of biomarkers in sequential biopsies of patients with chronic wounds receiving simultaneous acute wounds: a genetic, histological, and noninvasive imaging study.

Chronic wounds are common and lead to significant patient morbidity. A better understanding of their pathogenesis and relevant biomarkers are required. We compared acute and chronic wounds in the same individual using noninvasive imaging including spectrophotometric intracutaneous analysis (SIAscopy) and full‐field laser perfusion imaging. Gene expression analysis was also performed on sequential biopsies. Whole genome gene expression microarray analysis (44k), quantitative polymerase chain reaction, and immunohistochemistry were carried out to determine gene expression levels in tissue biopsies. Fifteen Caucasian patients with chronic venous ulcers had biopsies of the wound edges and simultaneously had an acute wound created on their upper arm on days 0, 7, and 14. SIAscopy revealed increased levels of melanin (p < 0.001), reduced levels of collagen (p < 0.001), and hemoglobin (p = 0.022) in chronic wounds. Microarray and subsequent quantitative polymerase chain reaction analysis confirmed an overall differential expression in acute and chronic wounds for several genes. Significantly higher levels of inhibin, beta A (INHBA) expression were confirmed in the dermis of chronic wounds (p < 0.05). Additionally, INHBA and thrombospondin 1 messenger RNA levels significantly correlated with SIAscopy measurements (p < 0.05). This unique study has showed aberrant expression of INHBA in chronic wounds using a sequential biopsy model of chronic vs. acute wounds in the same individual.

DNA copy number variations at chromosome 7p14.1 and chromosome 14q11.2 are associated with dupuytren's disease: potential role for MMP and Wnt signaling pathway.

Background: Dupuytrens disease is a common fibroproliferative disorder with an unknown etiology. Emerging evidence suggests a strong genetic component involved in the manifestation of the disease. This study aims to investigate the potential involvement of copy number variations in Dupuytrens disease pathogenesis. Methods: Array-based comparative genomic hybridization (NimbleGen Human CGH 2.1 M) was utilized to compare DNA from (1) nodules versus internal control (patients blood; n = 4) and (2) nodules (n = 4) versus external control (commercial reference DNA pooled from 10 donors). Analysis was carried out using Nexus 5.1 (BioDiscovery, El Segundo, Calif.) with the inclusion of additional results from previously published array-based comparative genomic hybridization. Copy number variations were considered to be common in Dupuytrens disease if the overlap was statistically significant and they were present in the majority (75 to 87.5 percent when compared with controls) of Dupuytrens disease nodules. The copy number variations loci were also compared with recently published genome wide-association studies. Common copy number variations were further validated using quantitative polymerase chain reaction. DNA from 25 Dupuytrens disease cases and 30 external controls were used in the quantitative polymerase chain reaction validation. In addition, gene expression was compared between Dupuytrens disease nodules and internal controls (transverse palmar fascia; n = 7). Results: Five common copy number variations, on chromosome 17q12, 1p31.1, 20p13, 7p14.1, and 14q11.2, were identified by array-based comparative genomic hybridization. Significantly higher copy numbers of copy number variations at chromosome 7p14.1 and 14q11.2 in Dupuytrens disease were confirmed in quantitative polymerase chain reaction validation. Matrix metalloproteinase-14 and secreted frizzled-related protein 4 (near a polymorphism recently associated with Dupuytrens disease) were significantly up-regulated in nodules. Conclusion: This study demonstrated an association between Dupuytrens disease and copy number variations at chromosomes 7p14.1 and 14q11.2.

Tumour Necrosis Factor-α Expression Is Associated with Increased Severity of Periprosthetic Breast Capsular Contracture

Background: The pathogenesis of periprosthetic capsular contracture following breast implant surgery is unclear. The aim of this study was to identify the expression of tumour necrosis factor-α (TNF-α), collagen type III α1 (COL3A1), transforming growth factor-β1 (TGF-β1) and connective tissue growth factor (CTGF) in different Baker grades of breast capsular contracture. Methods: Seven periprosthetic breast capsule specimens were collected from 6 patients. TNF-α, COL3A1, TGF-β1 and CTGF gene expression were analysed using real-time quantitative polymerase chain reaction. Immunohistolocalisation of TNF-α was performed on paraffin-embedded sections. Significant correlations were analysed using the Pearson correlation coefficient. Results: TNF-α expression was associated with increased Baker grade of capsular contracture (Pearson correlation, r = 0.558; p = 0.02). COL3A1 gene expression was reduced with increasing severity of contracture (r = –0.490; p = 0.05). There were no significant correlations between TGF-β1 and CTGF expression with Baker grade. Positive TNF-α staining in breast capsules was localised to fibroblasts, macrophages, and extracellularly close to the prosthesis. Conclusion: The increased expression of TNF-α may play a key role in the inflammatory response associated with capsular contracture. The corresponding decrease in COL3A1 may contribute to the change in capsular physical properties seen in capsular contracture.

Identification of molecular phenotypic descriptors of breast capsular contracture formation using informatics analysis of the whole genome transcriptome.

Breast capsular contracture formation following silicone implant augmentation/reconstruction is a common complication that remains poorly understood. The aim of this study was to identify potential biomarkers implicated in breast capsular contracture formation by using, for the first time, whole genome arrays. Biopsy samples were taken from 18 patients (23 breast capsules) with Baker Grade I–II (Control) and Baker Grade III–IV (Contracted). Whole genome microarrays were performed and six significantly dysregulated genes were selected for further validation with quantitative reverse transcriptase polymerase chain reaction and immunohistochemistry. Hematoxylin and eosin was also carried out to compare the histological characteristics of control and contracted samples. Microarray results showed that aggrecan, tissue inhibitor of metalloproteinase 4 (TIMP4), and tumor necrosis factor superfamily (ligand) member 11 were significantly down‐regulated in contracted capsules; while matrix metallopeptidase 12, serum amyloid A 1, and interleukin 8 (IL8) were significantly up‐regulated. The dysregulation of aggrecan, tumor necrosis factor superfamily (ligand) member 11, TIMP4, and IL8 was validated by quantitative reverse transcriptase polymerase chain reaction (p < 0.05). Immunohistochemistry confirmed an increased protein expression for IL8 and matrix metallopeptidase 12 in contracted capsules (p < 0.05), and decreased protein expression of TIMP4 (p < 0.05). This study has shown, for the first time, a number of unique biomarkers of significance in capsular contracture formation. IL8 and TIMP4 may serve as potential key diagnostic, therapeutic, and prognostic biomarkers in capsular contracture formation.