The myofibroblast, a key cell in normal and pathological tissue repair.

Myofibroblasts are characterized by their expression of α-smooth muscle actin, their enhanced contractility when compared to normal fibroblasts and their increased synthetic activity of extracellular matrix proteins. Myofibroblasts play an important role in normal tissue repair processes, particularly in the skin where they were first described. During normal tissue repair, they appear transiently and are then lost via apoptosis. However, the chronic presence and continued activity of myofibroblasts characterize many fibrotic pathologies, in the skin and internal organs including the liver, kidney and lung. More recently, it has become clear that myofibroblasts also play a role in many types of cancer as stromal or cancer-associated myofibroblast. The fact that myofibroblasts are now known to be key players in many pathologies makes understanding their functions, origin and the regulation of their differentiation important to enable them to be regulated in normal physiology and targeted in fibrosis, scarring and cancer.

Hypoxia in tissue repair and fibrosis.

Hypoxia and hypoxia signalling through the transcription factor hypoxia inducible factor-1 (HIF-1), play an important role in normal tissue repair processes. Tissue injury generally produces at least the transient loss of normal vascular perfusion and the resulting hypoxia induces the expression of many genes that allow the tissue to adapt to hypoxia, to start the repair process and, in time, to re-establish oxygen delivery to the tissue. In most cases, transient hypoxia and the activation of the HIF-1 pathway are beneficial and promote the repair process, producing tissue that might not perfectly reform its original architecture but that has its function substantially restored. However, in some cases of chronic injury, chronic hypoxia and pathological repair, the hypoxia pathway might be responsible for driving the process of fibrosis and can lead to excessive scarring and compromised organ function.

Siah2-deficient mice show impaired skin wound repair.

Hypoxia is associated with the dermal wound healing process and hypoxia signaling is presumed to be crucial for normal wound repair. The Siah2 ubiquitin ligase controls the abundance of hypoxia-inducible factor-1 alpha, and loss of Siah2 results in destabilization of hypoxia-inducible factor-1 alpha under hypoxia. Utilizing Siah2(-/-) mice we demonstrate that cutaneous wound healing is impaired in these mice. Wounds in Siah2(-/-) mice heal slower and are associated with delayed induction of myofibroblast infiltration and reduced collagen deposition. This coincides with delayed angiogenesis and reduced macrophage infiltration into the wounds of Siah2(-/-) mice. We furthermore demonstrate that primary Siah2(-/-) dermal fibroblasts have reduced migratory capacities and produce less collagen than wild-type fibroblasts. Additionally, Siah2(-/-) fibroblasts showed conserved responses to transforming growth factor-ß at the receptor level (pSmad 2C activation) but reduced responses downstream. Together, our data show, for the first time, that Siah2 is involved as a positive regulator in the wound healing response. Understanding the role of hypoxia signaling in tissue repair and fibrosis and interference with the hypoxia signaling pathway via regulation of Siah2 may provide new targets for clinical regulation of fibrosis and scarring.

Self-Assembled Peptide Habitats to Model Tumor Metastasis.

Metastatic tumours are complex ecosystems; a community of multiple cell types, including cancerous cells, fibroblasts, and immune cells that exist within a supportive and specific microenvironment. The interplay of these cells, together with tissue specific chemical, structural and temporal signals within a three-dimensional (3D) habitat, direct tumour cell behavior, a subtlety that can be easily lost in 2D tissue culture. Here, we investigate a significantly improved tool, consisting of a novel matrix of functionally programmed peptide sequences, self-assembled into a scaffold to enable the growth and the migration of multicellular lung tumour spheroids, as proof-of-concept. This 3D functional model aims to mimic the biological, chemical, and contextual cues of an in vivo tumor more closely than a typically used, unstructured hydrogel, allowing spatial and temporal activity modelling. This approach shows promise as a cancer model, enhancing current understandings of how tumours progress and spread over time within their microenvironment.

Explanting is an ex vivo model of renal epithelial-mesenchymal transition.

Recognised by their de novo expression of alpha-smooth muscle actin (SMA), recruitment of myofibroblasts is key to the pathogenesis of fibrosis in chronic kidney disease. Increasingly, we realise that epithelial-mesenchymal transition (EMT) may be an important source of these cells. In this study we describe a novel model of renal EMT. Rat kidney explants were finely diced on gelatin-coated Petri dishes and cultured in serum-supplemented media. Morphology and immunocytochemistry were used to identify mesenchymal (vimentin+, α-smooth muscle actin (SMA)+, desmin+), epithelial (cytokeratin+), and endothelial (RECA+) cells at various time points. Cell outgrowths were all epithelial in origin (cytokeratin+) at day 3. By day 10, 50 ± 12% (mean ± SE) of cytokeratin+ cells double-labelled for SMA, indicating EMT. Lectin staining established a proximal tubule origin. By day 17, cultures consisted only of myofibroblasts (SMA+/cytokeratin-). Explanting is a reproducible ex vivo model of EMT. The ability to modify this change in phenotype provides a useful tool to study the regulation and mechanisms of renal tubulointerstitial fibrosis.

Using in situ hybridisation to localize renal gene expression in tissue sections.

The basics of in situ hybridization have been widely applied to a diverse range of situations where we need to localize the distribution of nucleic acids. Advances in other molecular techniques such as the advent of gene microarrays has not diminished the significance of in situ hybridization, but rather highlight the importance of being able to identify the topology of gene expression. In situ hybridization offers a degree of precision that is unavailable with other molecular techniques. This chapter outlines techniques used to examine the spatial distribution of gene expression in the kidney using complementary RNA (cRNA) probes with both radioactive and non-radioactive labels.

Fibroblast differentiation in wound healing and fibrosis.

The contraction of granulation tissue from skin wounds was first described in the 1960s. Later it was discovered that during tissue repair, fibroblasts undergo a change in phenotype from their normal relatively quiescent state in which they are involved in slow turnover of the extracellular matrix, to a proliferative and contractile phenotype termed myofibroblasts. These cells show some of the phenotypic characteristics of smooth muscle cells and have been shown to contract in vitro. In the 1990s, a number of researchers in different fields showed that myofibroblasts are present during tissue repair or response to injury in a variety of other tissues, including the liver, kidney, and lung. During normal repair processes, the myofibroblastic cells are lost as repair resolves to form a scar. This cell loss is via apoptosis. In pathological fibroses, myofibroblasts persist in the tissue and are responsible for fibrosis via increased matrix synthesis and for contraction of the tissue. In many cases this expansion of the extracellular matrix impedes normal function of the organ. For this reason much interest has centered on the derivation of myofibroblasts and the factors that influence their differentiation, proliferation, extracellular matrix synthesis, and survival. Further understanding of how fibroblast differentiation and myofibroblast phenotype is controlled may provide valuable insights into future therapies that can control fibrosis and scarring.

Protection against reperfusion injury by 3',4'-dihydroxyflavonol in rat isolated hearts involves inhibition of phospholamban and JNK2.

BACKGROUND: Flavonols, including 3',4'-dihydroxyflavonol (DiOHF), reduce myocardial ischemia and reperfusion (I/R) injury but their mechanism remains uncertain. To better understand the mechanism of the cardioprotective actions of flavonols we investigated the effect of DiOHF on cardiac function and the activation of protective and injurious signalling kinases after I/R in rat isolated hearts. METHODS: We assessed the effect of global ischemia (20min) and reperfusion (5-30min) on cardiac function and injury in rat isolated, perfused hearts in the absence or presence of DiOHF (10µM) during reperfusion. Western blotting was used to assess changes in the phosphorylation state of kinases known to be involved in injury or protection. RESULTS: DiOHF improved cardiac contractility and reduced perfusion pressure and cell death in the isolated hearts. Phosphorylation of p38MAPK and CaMKII increased during ischemia with no further increase during reperfusion. Phosphorylation of other kinases increased during reperfusion. Phosphorylation of phospholamban (PLN) peaked at 5min of reperfusion whereas phosphorylation of Akt, Erk, STAT3 and JNK2 was highest after 30min. The presence of DiOHF during reperfusion significantly inhibited the activation of PLN and JNK without affecting phosphorylation of the protective kinases Erk1/2 and STAT3. Experiments in vitro demonstrated that DiOHF inhibited CaMKII by competing with ATP but not Ca2+/calmodulin. CONCLUSIONS: It is proposed that DiOHF confers protection against myocardial reperfusion injury by inhibiting CaMKII and subsequent PLN-induced leak of Ca2+ from the sarcoplasmic reticulum as well as by inhibiting JNK2 activation to reduce apoptosis.

Role of the phosphatidylinositol 3-kinase and mTOR pathways in the regulation of renal fibroblast function and differentiation.

Tubulointerstitial fibrosis is largely mediated by (myo)fibroblasts present in the interstitium. In this study, we investigated the role of mTOR and phosphatidylinositol 3-kinase in the regulation of fibroblast kinetics, fibroblast differentiation, and collagen synthesis. Rat renal fibroblasts were propagated from kidneys 3 days post-ureteric obstruction and specific inhibitors of mTOR (RAD) and phosphatidylinositol 3-kinase (LY294002) were used to examine the regulation of fibrogenesis. LY294002 but not RAD completely inhibited phosphorylation of Akt, while both inhibitors decreased phosphorylation of the S6 ribosomal protein. RAD and LY decreased foetal calf serum stimulated proliferation and DNA synthesis. In addition to their individual effects, treatment with both RAD and LY294002 decreased serum-induced fibroblast proliferation and DNA synthesis significantly more than either drug alone. TUNEL positive cells (apoptosis) in RAD and LY294002 treated groups were not different from control groups. In addition to their effect on proliferation, both inhibitors also reduced total collagen synthesis. Differentiation studies indicated an increase in alpha-smooth muscle actin expression relative to beta-actin (western blotting), with cytochemistry confirming that all doses of RAD and LY294002 increased the proportion of alpha-smooth muscle actin positive cells, and hence myofibroblasts. Effects were independent of cell toxicity. These results highlight the potential significance of PI3K and mTOR, in the regulation of renal (myo)fibroblast activity. The synergistic effects of LY and RAD on proliferation suggest that mTOR signalling involves pathways other than phosphatidylinositol 3-kinase. These results provide a novel insight into the mechanisms of fibroblast regulation during fibrogenesis.

Histochemical localization of apoptosis with in situ labeling of fragmented DNA.

Cell death by apoptosis is now recognized widely as an important constituent of cell turnover and disease pathology. Characterized by the cleavage of DNA into oligonucleosome-sized DNA fragments, end-labeling of fragmented DNA often is used as an in situ histological marker of apoptosis. The judicious and appropriate use of this technique therefore provides us with an important tool for assessing cell kinetics. Protocols for both terminal transferase-mediated UTP nick end-labeling, so-called TUNEL, and the combination of TUNEL with immunohistochemical staining are presented here, along with a discussion of its significance and interpretation.

Histochemical localization of cell proliferation using in situ hybridization for histone mRNA.

Monoclonal antibodies to proliferation associated antigens have long been used to histologically localize mitogenesis. However, techniques that distinguish cells in the synthetic or S phase have tended to rely on the in vivo incorporation of tritiated thymidine or thymidine analogs such as bromodeoxyuridine. The necessity to pulse with these labels before retrieving tissue means that they cannot be used in humans and are not available retrospectively. Measuring expression of histones serves as a useful adjunct to these techniques. As expression of histone proteins (H2A, H2B, H3, H4) are restricted to the synthetic phase of the cell cycle, hybridization for histone mRNA precisely distinguishes those cells in the S phase. Measuring their expression can easily be applied to the histological localization of proliferation, and can be used both prospectively and with archived tissue specimens. Several histone in situ hybridization probes and nonradioactive detection systems are now available commercially. A generalized protocol for their use in measuring in situ proliferation is provided in this chapter.

In situ hybridization using cRNA probes: isotopic and nonisotopic detection methods.

In this chapter we describe the use of cRNA (riboprobes) in the detection of gene expression in tissue sections. Riboprobes offer good sensitivity and allow the detection of low-level mRNA expression. In some cases, the use of radiolabeling is justified because this method is still sensitive. However, recent advances in nonisotopic detection methods mean that in some cases digoxigenin (DIG) or biotin labeling also may be sufficiently sensitive to detect mRNA expression in tissues of interest. The use of alkaline phosphatase conjugated anti-DIG antibodies improves the sensitivity of DIG detection over peroxidase systems, and the use of amplification systems based on biotinyl tyramide has improved the sensitivity of biotin labelled probe detection. Finally, it can be shown that low-level mRNA expression is easier to detect in frozen sections than in paraffin-embedded material, with a consequent loss in quality of morphology.

Skeletal myocytes are a source of interleukin-6 mRNA expression and protein release during contraction: evidence of fiber type specificity.

In this study, we aimed to determine whether skeletal muscle cells per se are a source of interleukin (IL)-6 during contraction and whether IL-6 production is fiber type specific. Muscle biopsy samples were collected from seven males before (PRE) and after (POST) completing 120 min of continuous bicycle ergometry. Biopsies were sectioned and analyzed for the following: IL-6 protein detected by immunohistochemistry (IHC), IL-6 mRNA content detected by in situ hybridization, fiber type measured by either IHC or myofibrillar ATPase activity stain, and glycogen content measured by periodic acid schiff (PAS) assay. Fibers were qualitatively categorized according to glycogen content to one of five groups (1-5), with 1 being very low (LOW) and 5 being very high (HIGH) glycogen. Total fluorescence (PRE vs. POST) and glycogen-dependent fluorescence (LOW vs. HIGH) of IL-6 protein were quantitated using Metamorph software. Total IL-6 protein was elevated from PRE to POST exercise (P<0.05). At PRE, IL-6 protein was evenly distributed across all fibers at low levels, consistent with glycogen distribution. At POST, IL-6 protein was greater (P<0.05) in HIGH compared with LOW glycogen fibers, which coincided with type 2 fibers. IL-6 mRNA was distributed peripherally in all fibers at PRE. At POST, however, IL-6 mRNA appeared predominantly in type 2 fibers, which also had higher glycogen content (P<0.05). These data demonstrate that myocytes per se are a source of IL-6 produced during contraction. Our data also suggest that type 2 fibers predominantly produce IL-6 during muscle contractile activity.

Quantitative proteomics of bronchoalveolar lavage fluid in lung adenocarcinoma.

BACKGROUND: The most commonly reported primary lung cancer subtype is adenocarcinoma, which is associated with a poor prognosis and short survival. Proteomic studies on human body fluids such as bronchoalveolar lavage fluid (BALF) have become essential methods for biomarker discovery, examination of tumor pathways and investigation of potential treatments. AIM: This study used quantitative proteomics to investigate the up-regulation of novel proteins in BALF from patients with primary lung adenocarcinoma in order to identify potential biomarkers. MATERIALS AND METHODS: BALF samples from individuals with and without primary lung adenocarcinoma were analyzed using liquid chromatography-mass spectrometry. RESULTS: One thousand and one hundred proteins were identified, 33 of which were found to be consistently overexpressed in all lung adenocarcinoma samples compared to non-cancer controls. A number of overexpressed proteins have been previously shown to be related to lung cancer progression including S100-A8, annexin A1, annexin A2, thymidine phosphorylase and transglutaminase 2. CONCLUSION: The overexpression of a number of specific proteins in BALF from patients with primary lung adenocarcinoma may be used as a potential biomarker for lung adenocarcinoma.

Cytoskeletal protein Flightless I inhibits apoptosis, enhances tumor cell invasion and promotes cutaneous squamous cell carcinoma progression.

Flightless I (Flii) is an actin remodeling protein that affects cellular processes including adhesion, proliferation and migration. In order to determine the role of Flii during carcinogenesis, squamous cell carcinomas (SCCs) were induced in Flii heterozygous (Flii+/-), wild-type and Flii overexpressing (FliiTg/Tg) mice by intradermal injection of 3-methylcholanthrene (MCA). Flii levels were further assessed in biopsies from human SCCs and the human SCC cell line (MET-1) was used to determine the effect of Flii on cellular invasion. Flii was highly expressed in human SCC biopsies particularly by the invading cells at the tumor edge. FliiTg/Tg mice developed large, aggressive SCCs in response to MCA. In contrast Flii+/- mice had significantly smaller tumors that were less invasive. Intradermal injection of Flii neutralizing antibodies during SCC initiation and progression significantly reduced the size of the tumors and, in vitro, decreased cellular sphere formation and invasion. Analysis of the tumors from the Flii overexpressing mice showed reduced caspase I and annexin V expression suggesting Flii may negatively regulate apoptosis within these tumors. These studies therefore suggest that Flii enhances SCC tumor progression by decreasing apoptosis and enhancing tumor cell invasion. Targeting Flii may be a potential strategy for reducing the severity of SCCs.

Fibroblasts and myofibroblasts in wound healing.

(Myo)fibroblasts are key players for maintaining skin homeostasis and for orchestrating physiological tissue repair. (Myo)fibroblasts are embedded in a sophisticated extracellular matrix (ECM) that they secrete, and a complex and interactive dialogue exists between (myo)fibroblasts and their microenvironment. In addition to the secretion of the ECM, (myo)fibroblasts, by secreting matrix metalloproteinases and tissue inhibitors of metalloproteinases, are able to remodel this ECM. (Myo)fibroblasts and their microenvironment form an evolving network during tissue repair, with reciprocal actions leading to cell differentiation, proliferation, quiescence, or apoptosis, and actions on growth factor bioavailability by binding, sequestration, and activation. In addition, the (myo)fibroblast phenotype is regulated by mechanical stresses to which they are subjected and thus by mechanical signaling. In pathological situations (excessive scarring or fibrosis), or during aging, this dialogue between the (myo)fibroblasts and their microenvironment may be altered or disrupted, leading to repair defects or to injuries with damaged and/or cosmetic skin alterations such as wrinkle development. The intimate dialogue between the (myo)fibroblasts and their microenvironment therefore represents a fascinating domain that must be better understood in order not only to characterize new therapeutic targets and drugs able to prevent or treat pathological developments but also to interfere with skin alterations observed during normal aging or premature aging induced by a deleterious environment.

Temporal variation in the deposition of different types of collagen within a porous biomaterial implant.

The deposition of new collagen in association with a medical implant has been studied using expanded polytetrafluoroethylene vascular replacement samples implanted subcutaneously in sheep, for up to 28 days. New type I collagen mRNA synthesis was followed by in situ hybridization, while the accumulation of new collagen types III, V, VI, XII, and XIV was followed by immunohistochemistry. All the collagen detected in the pores of the implant were newly deposited at various times after implantation and were not due to any pre-existing dermal collagen that may have been present around the implant. Collagen deposition was seen initially surrounding the implant and, with time, was seen to infiltrate within its pores. In situ hybridization showed that the majority of infiltrating cells had switched on mRNA that coded for type I collagen production. Histology showed that cellular infiltration increased with time, accompanied by increasing collagen deposition. The deposition of different collagen types happened at different rates. The type V and VI collagens preceded the major interstitial collagens in the newly deposited tissue, although at longer time points, detection of type V collagen appeared to decrease. After disruption of the interstitial collagens with enzyme, the "masked" type V collagen was clearly still visible by immunohistochemistry. Little type XII collagen could be seen within the porous mesh, although it was seen in the surrounding tissues. By contrast, type XIV was seen throughout the porous structure of the implanted mesh, with less being visible outside the material where type XII was more abundant.

Blood classical monocytes phenotype is not altered in primary non-small cell lung cancer.

AIM: To evaluate the M1 and M2 monocyte phenotype in patients with non-small cell lung cancer (NSCLC) compared to controls. Also, to examine the expression of Th1 and Th2 cytokines in plasma of NSCLC vs controls. METHODS: Freshly prepared peripheral blood mononuclear cells samples were obtained from patients with NSCLC (lung adenocarcinoma and squamous cell lung carcinoma) and from non-cancer controls. Flow cytometry was performed to investigate M1 and M2 phenotypes in peripheral monocytes (classical monocytes CD14+, CD45+ and CD16-) using conventional surface markers. Th1 and Th2 cytokine production was also analysed in the plasma using cytometric bead array technique. RESULTS: There were no significant difference in expression of M1 (HLA-DR) and/or M2 markers (CD163 and CD36) markers on classical monocytes in patients with NSCLC compared to non-cancer controls. Expression of CD11b, CD11c, CD71 and CD44 was also shown to be similar in patients with NSCLC compared to non-cancer controls. Th1 and Th2 cytokines [interleukin (IL)-1ß, IL-2, IL-4, IL-5, IL-8, IL-10, IL-12 (p70), tumor necrosis factor (TNF)-α, TNF-ß, and interferon-γ] analysis revealed no significant difference between patients with NSCLC and non-cancer controls. CONCLUSION: This study shows no alteration in peripheral monocyte phenotype in circulating classical monocytes in patients with NSCLC compared to non-cancer controls. No difference in Th1 and Th2 cytokine levels were noted in the plasma of these patients.

Hypoxia and hypoxia signaling in tissue repair and fibrosis.

Following injury, vascular damage results in the loss of perfusion and consequent low oxygen tension (hypoxia) which may be exacerbated by a rapid influx of inflammatory and mesenchymal cells with high metabolic demands for oxygen. Changes in systemic and cellular oxygen concentrations induce tightly regulated response pathways that attempt to restore oxygen supply to cells and modulate cell function in hypoxic conditions. Most of these responses occur through the induction of the transcription factor hypoxia-inducible factor-1 (HIF-1) which regulates many processes needed for tissue repair during ischemia in the damaged tissue. HIF-1 transcriptionally upregulates expression of metabolic proteins (GLUT-1), adhesion proteins (integrins), soluble growth factors (TGF-ß and VEGF), and extracellular matrix components (type I collagen and fibronectin), which enhance the repair process. For these reasons, HIF-1 is viewed as a positive regulator of wound healing and a potential regulator of organ repair and tissue fibrosis. Understanding the complex role of hypoxia in the loss of function in scarring tissues and biology of chronic wound, and organ repair will aid in the development of pharmaceutical agents that can redress the detrimental outcomes often seen in repair and scarring.

The myofibroblast, multiple origins for major roles in normal and pathological tissue repair.

Myofibroblasts differentiate, invade and repair injured tissues by secreting and organizing the extracellular matrix and by developing contractile forces. When tissues are damaged, tissue homeostasis must be re-established, and repair mechanisms have to rapidly provide harmonious mechanical tissue organization, a process essentially supported by (myo)fibroblasts. Under physiological conditions, the secretory and contractile activities of myofibroblasts are terminated when the repair is complete (scar formation) but the functionality of the tissue is only rarely perfectly restored. At the end of the normal repair process, myofibroblasts disappear by apoptosis but in pathological situations, myofibroblasts likely remain leading to excessive scarring. Myofibroblasts originate from different precursor cells, the major contribution being from local recruitment of connective tissue fibroblasts. However, local mesenchymal stem cells, bone marrow-derived mesenchymal stem cells and cells derived from an epithelial-mesenchymal transition process, may represent alternative sources of myofibroblasts when local fibroblasts are not able to satisfy the requirement for these cells during repair. These diverse cell types probably contribute to the appearance of myofibroblast subpopulations which show specific biological properties and which are important to understand in order to develop new therapeutic strategies for treatment of fibrotic and scarring diseases.