IH occurs as a result of fascial tissue deficiency due to impairment in surgical wound healing following a laparotomy [18]. Pathological evaluation of tissue from IH patients discloses cellular and extracellular derangements in the abdominal wall tissue [19]. Disturbance in collagen homeostasis has been previously incriminated in the development of IH. Our study supports the concept that abnormal/altered fibroblast population results in the synthesis of abnormal ECM components leading to the development of IH following a laparotomy [13]. The exact mechanism of the selection of altered fibroblast phenotypes is unknown; however, it is believed that the loss of mechanical integrity of abdominal wall due to the incision somehow signals the fibroblasts to attain a pathological phenotype resulting in defective collagen production [19] [20]. Since the tissue fibroblasts are associated with ECM homeostasis, alterations in the fibroblast phenotypes signify a possible mechanism underlying IH formation. The identification and characterization of pathological fibroblasts in the IH tissues should pave the way for a better understanding the IH pathology.
In this study, the histological architecture of the hernia tissues was altered considerably when compared with the control suggesting that the persistence of ECM disorganization is involved in IH pathology. A shift in nuclear morphology from linear to oval shape was observed in fibroblast cells of IH tissues signifying the existence of altered phenotypes. It has been reported that the oval, spindle and/or stellate shaped cells with round or oval nuclei represent active (proliferative) fibroblasts which reflect ECM pathology [21]. Based on the nuclear morphology, the proliferative fibroblasts were predominant in HRT and HST. On the other hand, the control tissues displayed elongated wavy nuclei representing mature/inactive fibroblasts which were surrounded by intact ECM [21] [22]. Moreover, the fibroblasts exist in the tissues as heterogeneous phenotypes with various subpopulations having distinct functions other than ECM homeostasis such as immune accessory cells, wound healing, inflammation and tissue regeneration [21].
The current strategies are limited to define the heterogeneity of fibroblasts under physiological and pathological conditions and to understand their complex role in IH pathology. Our approach was to examine and compare the fibroblast phenotypes by assessing the expression of fibroblast specific biomarkers in IH and normal abdominal wall tissues. The FSP1 (also known as S100A4 and is a specific biomarker for fibroblasts) has a significant role in regulating epithelial to mesenchymal transition, apart from its role in fibrosis [23] [24] [25]. The increased expression of FSP1 in HST can be correlated with fatty infiltration as observed by histology. The increased expression of FSP1 with concomitantly increased adipose tissue infiltration suggesting the possible role of fibroblasts as adipocyte precursor. However, further investigations are warranted to validate the adipogenic function of fibroblasts in the IH tissues.
CD34 is a transmembrane glycoprotein which marks hematopoietic progenitor cells and mesenchymal stem cells (MSCs) [26]. As the expression of CD34 was confined to extravascular tissues, the possibility of endothelial cell lineages is minimal in the studied specimen. Also, CD34 is a biomarker for undifferentiated fibroblasts that are critical in tissue remodeling [27]. Since MSCs and fibroblasts share mesenchymal origin and specific biomarkers, further characterization are warranted to distinguish these cell population. The progressive loss of CD34 paves the way in the induction of α-SMA + myofibroblasts [28]. CD34 + cells were increased in HST when compared to the control, which was in accordance with the histological findings. This suggests that the proliferation of undifferentiated fibroblasts is an obstacle in the trans-differentiation of myofibroblasts resulting in abnormal wound healing. Also, the undifferentiated fibroblasts secrete incomplete ECM and result in disorganization as evident in the histology [21]. The similar expression profile of CD34 in HRT and FT reveals the presence of matured tissue suggesting that the IH pathology nucleates at HST by depositing abnormal ECM which eventually leads to form IH in an unknown mechanism and warrants further investigation.
The α-SMA has been considered to be a pivotal biomarker for myofibroblasts, which are contractile phenotype of fibroblasts mainly associated with wound healing response; especially in the process of wound contraction [29] [30]. The decreased expression of α-SMA in the HST and HRT shows the impaired wound healing phase or wound progression suggesting the persistence of pathology or dysfunction of myofibroblasts. Moreover, the correlation of α-SMA with the status of wound healing and severity of IH pathology is warranted deciphering its role in IH formation. Moreover, multiple cell phenotypes including vascular smooth muscle cells, pericytes and fibroblasts express α-SMA and we performed quantification of the fluorescence intensity for total α-SMA. In addition, the blood vessels in tissue sections displayed upregulation of α-SMA; however, the exclusion of blood vessels from the analysis was difficult owing to their increased density. Interestingly, extra vascular tissues displayed the immunopositivity for α-SMA suggesting the α-SMA-positive fibroblast phenotypes as predominantly evident in HST and HRT which provides qualitative understanding regarding the expression status in α-SMA extra vascular tissues. However, co-staining with extravascular biomarkers is warranted to distinguish fibroblasts from cells of vascular lineage. Based on these observations, it is logical that the trans-differentiation signaling of fibroblasts to myofibroblasts is impaired in HRT tissues resulting in abnormal wound healing and IH formation. However, the exact mechanisms underlying the trans-differentiation of myofibroblasts in IH tissues are unknown and warrant further investigation.
CTGF has been identified as a fibrogenic factor which co-expresses with transforming growth factor-β (TGF-β) and triggers fibroblast proliferation, migration, and ECM synthesis. Basal level of CTGF expression has been reported in normal tissues whereas CTGF level increases in the fibrotic tissues [31]. Reactive oxygen species (ROS) and oxidative stress are well known triggers for CTGF expression; however the involvement of ROS in IH pathology has not been studied [32] [33]. Even though the actual role of CTGF in IH pathology is not clear the increased levels of CTGF in HRT and HST suggest a healing response elicited by the surviving tissue. However, CTGF-silenced animal models of IH could be helpful to elucidate the mechanism of CTGF action in IH pathology.Cadherin-11 is engaged in the release of pro-inflammatory signals such as IL-6, TNF-α and IL-1β from the fibroblasts of injured tissues [34] [35]. In addition, cadherin-11 is associated with the expression of various ECM components including collagen and elastin and regulates the mechanical integrity of the tissue and wound healing responses[36]. Despite significant upregulation of cadherin-11 in HRT and HST, the functional role of cadherin-11 in the inflammation and ECM damage associated with IH are largely unknown and requires more attention.
The present study focused on the examination of the expression of biomarkers of fibroblasts and to define their phenotypes based on their expression in IH. The biomarkers chosen for the current study were based on their characteristic expression on fibroblast subpopulations and displayed considerable alterations between HRT and HST and the respective controls FT and PT suggesting a possible association with IH pathology. However, the expression status of these biomarkers has not been established in post-laparotomy IH formation which requires further research to unveil the molecular events leading to IH formation. Moreover, the information regarding the characterization of abnormal fibroblast population in IH tissues based on these biomarkers is unavailable. To our knowledge, this is the first study focusing on phenotyping fibroblast in IH formation and the overall findings are displayed in Fig. 7. Our major findings revealed differential expression of these biomarkers in HST and HRT (the major susceptible sites for IH formation) suggesting the existence of diverse fibroblast phenotypes in these tissues which in turn reflects the difference in molecular pathology. However, the exact molecular mechanism underlying the regulation of diverse fibroblast phenotypes based on the biomarker expression warrants further investigation.
The present study has several limitations including the unavailability of specific biomarker for fibroblasts that limits the co-localization studies. Moreover, the specificity of the biomarkers is questionable since they might be expressed in other cell types including immune cells and stem cells. Hence, proper definition of IH fibroblasts based on specific biomarker panel is warranted. In addition, the cellular composition in HST and HRT has not been established and the phenotype of extra fibroblasts warrants further attention. Other limitations include the lack of co-expression studies of the biomarkers with other cell types such as MSCs, smaller size of control tissues, relatively small number of patient/control tissue specimen, variability among the patients and controls, and lack of in vitro validation. Due to the limited availability of control specimen and smaller size of specimen, it was not possible to perform PCR and western blot analysis. However, the present study utilized MFI-based semi-quantitative fold-change to represent the expression status of biomarkers. In addition, the present study mainly relied on the immunopathology and further investigation based on proteomics, genomics and metabolomics are warranted. Nevertheless, the findings from this study strongly suggest that the heterogeneity of the fibroblast population could play a critical role in the development of IH. Understanding the mechanism underlying the phenotype switch of these fibroblasts would open opportunities to develop novel strategies to prevent the development of IH following a laparotomy.