Type V Collagen in Scar Tissue Regulates the Size of Scar after Heart Injury.

Scar tissue size following myocardial infarction is an independent predictor of cardiovascular outcomes, yet little is known about factors regulating scar size. We demonstrate that collagen V, a minor constituent of heart scars, regulates the size of heart scars after ischemic injury. Depletion of collagen V led to a paradoxical increase in post-infarction scar size with worsening of heart function. A systems genetics approach across 100 in-bred strains of mice demonstrated that collagen V is a critical driver of postinjury heart function. We show that collagen V deficiency alters the mechanical properties of scar tissue, and altered reciprocal feedback between matrix and cells induces expression of mechanosensitive integrins that drive fibroblast activation and increase scar size. Cilengitide, an inhibitor of specific integrins, rescues the phenotype of increased post-injury scarring in collagen-V-deficient mice. These observations demonstrate that collagen V regulates scar size in an integrin-dependent manner.

Collagen microarchitecture mechanically controls myofibroblast differentiation.

Altered microarchitecture of collagen type I is a hallmark of wound healing and cancer that is commonly attributed to myofibroblasts. However, it remains unknown which effect collagen microarchitecture has on myofibroblast differentiation. Here, we combined experimental and computational approaches to investigate the hypothesis that the microarchitecture of fibrillar collagen networks mechanically regulates myofibroblast differentiation of adipose stromal cells (ASCs) independent of bulk stiffness. Collagen gels with controlled fiber thickness and pore size were microfabricated by adjusting the gelation temperature while keeping their concentration constant. Rheological characterization and simulation data indicated that networks with thicker fibers and larger pores exhibited increased strain-stiffening relative to networks with thinner fibers and smaller pores. Accordingly, ASCs cultured in scaffolds with thicker fibers were more contractile, expressed myofibroblast markers, and deposited more extended fibronectin fibers. Consistent with elevated myofibroblast differentiation, ASCs in scaffolds with thicker fibers exhibited a more proangiogenic phenotype that promoted endothelial sprouting in a contractility-dependent manner. Our findings suggest that changes of collagen microarchitecture regulate myofibroblast differentiation and fibrosis independent of collagen quantity and bulk stiffness by locally modulating cellular mechanosignaling. These findings have implications for regenerative medicine and anticancer treatments.

Connexin43 is differentially distributed within renal vasculature and mediates profibrotic differentiation in medullary fibroblasts.

Connexins (Cxs) form gap junctions for intercellular exchange of inorganic ions and messenger molecules. In the kidney, Cxs play essential roles within its compartments, but data on the precise cellular localization and cell type-related function of their isoforms are scarce. We tested whether Cx43 distribution is restricted to vascular and interstitial cells and whether medullary fibroblasts express Cx43 to coordinate profibrotic signaling. Confocal immunofluorescence techniques, ultrastructural labeling, and functional experiments in cell culture were performed. Cx43 was chiefly expressed in the vasculature but was absent from tubular epithelia. All arterial, arteriolar, and lymphatic endothelia showed continuous Cx43 signal along their borders. In the inner medulla, only the interstitium showed Cx43 signals, which were assigned to fibroblasts and their processes. Cultured Cx43-expressing medullary fibroblasts served to study the role of gap junctions in a profibrotic context. In a dye spreading assay, Cx43-sensitive diffusion of Lucifer yellow was dependent on gap junctional passage. The addition of transforming growth factor-ß1 (5 ng/mL for 48 h) activated Cx43 biosynthesis and caused Cx43-sensitive transformation of the fibroblasts into a myofibroblast phenotype. This suggested that Cx43 gap junctional channels enable the coordination of profibrotic signaling between cells of the medullary interstitium. In summary, we demonstrate the presence of Cx43-expressing gap junctions within the two major renal compartments, the vasculature and interstitium. Endothelial Cx43 likely provides functions of an earlier-defined "electrical syncytium" within the vascular wall. Additionally, Cx43 facilitates profibrotic signaling between medullary interstitial fibroblasts.

Epigallocatechin-3-gallate increases autophagic activity attenuating TGF-ß1-induced transformation of human Tenon's fibroblasts.

We previously found that epigallocatechin-3-gallate (EGCG) could inhibit the myofibroblast transformation of human Tenon's fibroblasts, however, the underlying mechanism remained unclear. We therefore investigated whether the autophagic regulation involved in the anti-fibrotic function of EGCG. The fibroblasts were subjected to transforming growth factor beta-1 (TGF-ß1) induction followed by EGCG treatments. The autophagic flux was examined by transmission electron microscopy and autophagic flux analysis. The levels of autophagy-related proteins (LC3ß and p62) and alpha-smooth muscle actin (α-SMA) were measured by Western blot and immunofluorescence. Results showed that TGF-ß1 partially inhibited the autophagic function of Tenon's fibroblasts. But this inhibition effect was rescued by LY2157299, a TGF-ßR1 selective inhibitor. Compared with the cells treated with TGF-ß1 alone, EGCG treatments increased the amount of autophagosomes and autolysosomes, evaluated the ratio of LC3-II to LC3-I and decreased p62 level. Our results indicated that EGCG could recover the activity of autophagy in the TGF-ß1-treated cells. Moreover, treatments with EGCG significantly decreased the α-SMA expression. Taken together, these findings revealed that autophagic regulation involved in the action of EGCG against TGF-ß1-induced transformation of Tenon's fibroblasts. Through increasing intracellular autophagy, EGCG could be a potential anti-fibrotic reagent for preventing subconjunctival fibrosis after glaucoma filtration surgery.

Scleroderma-like Impairment in the Network of Telocytes/CD34+ Stromal Cells in the Experimental Mouse Model of Bleomycin-Induced Dermal Fibrosis.

Considerable evidence accumulated over the past decade supports that telocytes (TCs)/CD34+ stromal cells represent an exclusive type of interstitial cells identifiable by transmission electron microscopy (TEM) or immunohistochemistry in various organs of the human body, including the skin. By means of their characteristic cellular extensions (telopodes), dermal TCs are arranged in networks intermingled with a multitude of neighboring cells and, hence, they are thought to contribute to skin homeostasis through both intercellular contacts and releasing extracellular vesicles. In this context, fibrotic skin lesions from patients with systemic sclerosis (SSc, scleroderma) appear to be characterized by a disruption of the dermal network of TCs, which has been ascribed to either cell degenerative processes or possible transformation into profibrotic myofibroblasts. In the present study, we utilized the well-established mouse model of bleomycin-induced scleroderma to gain further insights into the TC alterations found in cutaneous fibrosis. CD34 immunofluorescence revealed a severe impairment in the dermal network of TCs/CD34+ stromal cells in bleomycin-treated mice. CD31/CD34 double immunofluorescence confirmed that CD31-/CD34+ TC counts were greatly reduced in the skin of bleomycin-treated mice compared with control mice. Ultrastructural signs of TC injury were detected in the skin of bleomycin-treated mice by TEM. The analyses of skin samples from mice treated with bleomycin for different times by either TEM or double immunostaining and immunoblotting for the CD34/α-SMA antigens collectively suggested that, although a few TCs may transition to α-SMA+ myofibroblasts in the early disease stage, most of these cells rather undergo degeneration, and then are lost. Taken together, our data demonstrate that TC changes in the skin of bleomycin-treated mice mimic very closely those observed in human SSc skin, which makes this experimental model a suitable tool to (i) unravel the pathological mechanisms underlying TC damage and (ii) clarify the possible contribution of the TC loss to the development/progression of dermal fibrosis. In perspective, these findings may have important implications in the field of skin regenerative medicine.

Ultrastructural characterization of cells in the tibial stump of ruptured human anterior cruciate ligament, their changes and significance with duration of injury.

Fibroblasts and myofibroblasts have been known to be present in both ruptured and intact human anterior cruciate ligament (ACL), and although their relevant histology and immunochemistry have been studied in the past, ultrastructural features of these cells are largely lacking. Therefore, we aim to characterise the ultrastructural details of these cells with the help of transmission electron microscopy (TEM) and to study the changes and their significance with duration of injury. Samples from 60 ruptured human ACL undergoing surgery were obtained and categorised according to duration of injury and observed under TEM with main focus on the following ultrastructural features: cellular morphology, presence of rough endoplasmic reticulum, Golgi apparatus, lamina, myofilaments, and presence of myofibroblasts. These features were further correlated with the duration of injury and association, if any, determined using appropriate statistical analysis. A total of 54 male and 6 female patients with mean duration of the injury of 23.01 ± 26.09 weeks (2-108 weeks) were included in the study and categorised into five groups based on duration of injury as follows: I (< 6 weeks), II (7-12 weeks), III (13-20 weeks), IV (21-50 weeks) and V (> 50 weeks). There was a significant association between the above-mentioned ultrastructural features and the duration of injury (p < 0.05) except for the presence of ovoid fibroblast cells (p = 0.53). Furthermore, number of myofibroblasts and cells with Golgi apparatus and rough endoplasmic reticulum was seen to peak at 13-20 weeks following injury. We describe ultrastructural features of fibroblast of different morphology along with myofibroblasts in the ligaments following injury, the changes in which might have a potential bearing on ligament healing.

Myofibroblastic lesions in the oral cavity: Immunohistochemical and ultrastructural analysis.

OBJECTIVE: To immunohistochemically characterize a group of oral myofibroblastic lesions (MLs) and to evaluate the ultrastructural features of myofibroblasts. MATERIAL AND METHODS: Using a tissue microarray technique (TMA), cases of myofibroma (MF), of nodular fasciitis (NF), of desmoplastic fibroma (DF), and of myofibroblastic sarcoma (MS) from the Universidad Autónoma Metropolitana Xochimilco, and a Private Oral Pathology Service in Mexico City were stained with antibodies against alpha-smooth muscle actin (α-SMA), H-caldesmon, vimentin, desmin, ß-catenin, CD34, anaplastic lymphoma protein kinase (ALK-1), and Ki-67. RESULTS: Nineteen of the 22 MF cases, 2/5 of the NF cases, 1/10 of the DF cases, and 1/2 of the MS cases were positive for α-SMA. 1/2 of the MS cases were positive for desmin; 6/10 of the DF cases were positive for ß-catenin, and 2 of the MF cases were positive for ALK-1. All of the MLs were positive for vimentin and negative for H-caldesmon and CD-34. The Ki-67 labeling index in all of the 8/22 MF, 3/5 NF, and 2/2 MS cases was ≥10%. For all of the MLs evaluated, ultrastructural analysis revealed spindle-shaped cells containing endoplasmic reticulum and peripheral actin filament bundles. CONCLUSION: In certain myofibroblastic lesions, the use of auxiliary techniques (such as immunohistochemistry) can be critical for differential diagnosis.

Pharmacological inhibition of autophagy by 3-MA attenuates hyperuricemic nephropathy.

Autophagy has been identified as a cellular process of bulk degradation of cytoplasmic components and its persistent activation is critically involved in the renal damage induced by ureteral obstruction. However, the role and underlying mechanisms of autophagy in hyperuricemic nephropathy (HN) remain unknown. In the present study, we observed that inhibition of autophagy by 3-methyladenine (3-MA) abolished uric acid-induced differentiation of renal fibroblasts to myofibroblasts and activation of transforming growth factor-ß1 (TGF-ß1), epidermal growth factor receptor (EGFR), and Wnt signaling pathways in cultured renal interstitial fibroblasts. Treatment with 3-MA also abrogated the development of HN in vivo as evidenced by improving renal function, preserving renal tissue architecture, reducing the number of autophagic vacuoles, and decreasing microalbuminuria. Moreover, 3-MA was effective in attenuating renal deposition of extracellular matrix (ECM) proteins and expression of α-smooth muscle actin (α-SMA) and reducing renal epithelial cells arrested at the G2/M phase of cell cycle. Injury to the kidney resulted in increased expression of TGF-ß1 and TGFß receptor I, phosphorylation of Smad3 and TGF-ß-activated kinase 1 (TAK1), and activation of multiple cell signaling pathways associated with renal fibrogenesis, including Wnt, Notch, EGFR, and nuclear factor-κB (NF-κB). 3-MA treatment remarkably inhibited all these responses. In addition, 3-MA effectively suppressed infiltration of macrophages and lymphocytes as well as release of multiple profibrogenic cytokines/chemokines in the injured kidney. Collectively, these findings indicate that hyperuricemia-induced autophagy is critically involved in the activation of renal fibroblasts and development of renal fibrosis and suggest that inhibition of autophagy may represent a potential therapeutic strategy for HN.

Deletion of CD28 Co-stimulatory Signals Exacerbates Left Ventricular Remodeling and Increases Cardiac Rupture After Myocardial Infarction.

BACKGROUND: Inflammatory responses, especially by CD4(+)T cells activated by dendritic cells, are known to be important in the pathophysiology of cardiac repair after myocardial infarction (MI). Although co-stimulatory signals through B7 (CD80/86) and CD28 are necessary for CD4(+)T cell activation and survival, the roles of these signals in cardiac repair after MI are still unclear. METHODS AND RESULTS: C57BL/6 (Control) mice and CD28 knockout (CD28KO) mice were subjected to left coronary artery permanent ligation. The ratio of death by cardiac rupture within 5 days after MI was significantly higher in CD28KO mice compared with Control mice. Although there were no significant differences in the infarct size between the 2 groups, left ventricular end-diastolic and end-systolic diameters were significantly increased, and fractional shortening was significantly decreased in CD28KO mice compared with Control mice. Electron microscopic observation revealed that the extent of extracellular collagen fiber was significantly decreased in CD28KO mice compared with Control mice. The number of α-smooth muscle actin-positive myofibroblasts was significantly decreased, and matrix metalloproteinase-9 activity and the mRNA expression of interleukin-1ß were significantly increased in CD28KO mice compared with Control mice. CONCLUSIONS: Deletion of CD28 co-stimulatory signals exacerbates left ventricular remodeling and increases cardiac rupture after MI through prolongation of the inflammatory period and reduction of collagen fiber in the infarct scars. (Circ J 2016; 80: 1971-1979).

Telocytes of Fascial Structures.

Currently, the exact role of telocytes within fascial structures is unknown. The morphology, distribution and behaviour of fascial telocytes as well as the mutual relationship between telocytes and other cellular fascia constituents should be definitely a subject of further studies. It will contribute to better understanding of the role of the fascial system in health and diseases, may shed light on the regeneration potential of these tissues and may help to find targets for future treatments for locomotor disorders, including fascial diseases. Last but not least, confirmation of the presence of telocytes within fascia may contribute to optimise the use of fascia as a graft material.

Spotlight on the three main hepatic fibrogenic cells in HCV-infected patients: Multiple immunofluorescence and ultrastructure study.

The present work deals with the simultaneous ultrastructure and triple immunofluorescence study of the three main hepatic fibrogenic cells, hepatic stellate cell, myofibroblast (MF), and fibroblast, in a group of hepatitis C virus (HCV) RNA positive patients, as their exact interrelation behavior in vivo with the progress of hepatic fibrosis is still inadequate. In this study, for the first time, cells having the morphological characteristic of MF and not bone marrow fibrocytes were revealed in liver portal vessels. This necessitates the reevaluation of the available knowledge concerning bone marrow fibrocyte. Also, the distribution, cellular interrelations, and the fate of MF were highlighted.

Astaxanthin prevents pulmonary fibrosis by promoting myofibroblast apoptosis dependent on Drp1-mediated mitochondrial fission.

Promotion of myofibroblast apoptosis is a potential therapeutic strategy for pulmonary fibrosis. This study investigated the antifibrotic effect of astaxanthin on the promotion of myofibroblast apoptosis based on dynamin-related protein-1 (Drp1)-mediated mitochondrial fission in vivo and in vitro. Results showed that astaxanthin can inhibit lung parenchymal distortion and collagen deposition, as well as promote myofibroblast apoptosis. Astaxanthin demonstrated pro-apoptotic function in myofibroblasts by contributing to mitochondrial fission, thereby leading to apoptosis by increasing the Drp1 expression and enhancing Drp1 translocation into the mitochondria. Two specific siRNAs were used to demonstrate that Drp1 is necessary to promote astaxanthin-induced mitochondrial fission and apoptosis in myofibroblasts. Drp1-associated genes, such as Bcl-2-associated X protein, cytochrome c, tumour suppressor gene p53 and p53-up-regulated modulator of apoptosis, were highly up-regulated in the astaxanthin group compared with those in the sham group. This study revealed that astaxanthin can prevent pulmonary fibrosis by promoting myofibroblast apoptosis through a Drp1-dependent molecular pathway. Furthermore, astaxanthin provides a potential therapeutic value in pulmonary fibrosis treatment.

The "myofibroblast" that is omnipresent in pathology and key to the EMT concepts does not actually exist, since normal fibroblasts contain stress fibril organelles (SMA bundles with dense bodies) variably detected by TEM and IHC: conclusions by a diagnostic pathologist with decades of ultrastructural experience.

The so-called "enigmatic" unique "myofibroblast" has been erroneously substituted for virtually all things fibroblastic in soft tissue pathology and believed to be the ultimate fibrogenic cell. It is also internationally considered to be the mesenchymal cell in un-proven post-natal EMT, EMT organ/tissue fibrosis, and the assumption that EMT/MET is key to carcinoma/adenocarcinoma invasion and metastasis. However, no such cell exists, having been mistaken for our normal ubiquitous fibrogenic fibroblasts that contain peripheral bundles of actin (SMA) with dense bodies, i.e. stress fibril (SF) organelles variably detectable by TEM and SMA IHC, depending on the degree of activation. The only detectable features distinguishing what are erroneously believed to be two unique fibrogenic spindle cells are the SF. Is the variable detection of SF/SMA in fibroblastic and non-fibroblastic lesions significant? Carcinosarcomas are not bi-phasic malignancies or proof of EMT/MET. What does it mean that the fibroblasts of so-called "carcinoma-associated fibroblasts (CAF)" are not "myofibroblasts"? The true myofibroblast is the ultrastructurally and functionally unique, terminally-differentiated, pathognomonic cell of physiologic wound-healing, which unfortunately has been confused with the activated fibroblast. This study fails to demonstrate any ultrastructural evidence that either normal epithelial (EMT) or carcinoma/adenocarcinoma cells can undergo reversible transition into mesenchymal cells (EMT/MET) under any circumstances. The SF/SMA-positive fibrogenic cell in organ/tissue fibrosis is the genetically up-regulated, activated fibroblast, which has no relationship to EMT. Are any of the innumerable biochemical factors/elements considered to be associated with this non-existent cell and its related processes related to the activated fibroblast? The conclusions are based on review of every electron micrograph taken during a 40-year career in diagnostic and research ultrastructural pathology, and by confirming that the published TEM figures of so-called "myofibroblasts", are actually of fibroblasts.

Localization and interaction of genistein with model membranes formed with dipalmitoylphosphatidylcholine (DPPC).

The effect of genistein on the liposomes formed with dipalmitoylphosphatidylcholine was studied with the application of Fourier-transform infrared spectroscopy, nuclear magnetic resonance (1H NMR) and electron paramagnetic resonance techniques. Membranous structures organization of human skin fibroblasts and colon myofibroblasts was also examined using fluorescence and electron microscopy. The strongest rigidifying effect of genistein with respect to polar head groups was concluded on the basis of the effect of the flavonoid on the shape of NMR lines attributed to -N+(CH3)3 groups. The rigidifying effect of genistein with respect to the hydrophobic core of lipid membranes was also concluded from the genistein-dependent broadening of the NMR lines assigned to -CH2 groups and terminal -CH3 groups of alkyl chains. EPR data supported ordering effect of genistein of the hydrophobic core in the liquid-crystalline phase (Lalpha). The analysis of the FTIR spectra of the two-component liposomes showed that genistein incorporates into DPPC membranes via hydrogen bonding between the lipid polar head groups in the C-O-P-O-C segment and its hydroxyl groups. Both fluorescence microscopy and ultrastructural observation revealed changes in membranous structures organization as aftermath of genistein treatment. In conclusion, genistein localized within membranes changes the properties of membrane that can be followed by the changes inside cells being crucial for pharmacological activity of genistein used in cancer or other disease treatment.

Myofibroblastic differentiation of stromal cells in giant cell tumor of bone: an immunohistochemical and ultrastructural study.

The nature of the mononuclear stromal cells (MSCs) in giant cell tumor of bone (GCTB) has not been thoroughly investigated. The purpose of this study was to evaluate the degree and significance of myofibroblastic differentiation in 18 cases of GCTB by immunohistochemistry (IH) and/or electron microscopy (EM). All immunostained cases were found positive for smooth muscle actin (SMA) and/or muscle specific actin (MSA), most in 1-33% of the MSCs. Ultrastructurally, most MSCs were fibroblasts, and a significant number of cells displayed myofibroblastic differentiation. Myofibroblasts are an important component of MSCs in GCTB. The myofibroblastic population may be responsible in part for the production of matrix metalloproteinases (MMPs), which probably play a role in bone destruction, tumor aggression, and recurrence.

Comparison of the ultrastructural and immunophenotypic characteristics of human umbilical cord-derived mesenchymal stromal cells and in situ cells in Wharton's jelly.

The umbilical cord contains mucinous connective tissue, called Wharton's jelly. It consists of stromal cells, collagen fibers, and amorphous ground substances composed of proteoglycan. Recently, these stromal cells have been redefined as a new cell therapy source, named human umbilical cord-derived mesenchymal stromal cells (hUCMSCs). However, there are few studies on the ultrastructural features and immune-phenotypic characteristics of isolated hUCMSCs and comparisons with the cells found in original cord tissues. In this study, the authors describe and compare the phenotypic characteristics of hUCMSCs with cells in the umbilical cord in order to know the kinds of cells and ultrastructural changes. Isolated hUCMSCs showed similar ultrastructure with few structural differences from in situ stromal cells, and they are relatively homogenous and well-developed mesenchymal cells that demonstrate a myofibroblastic phenotype.

Ultrastructural assessment of the differentiation potential of human multipotent mesenchymal stromal cells.

Mesenchymal stromal (stem) cells (MSCs) are defined by plastic adherent growth, multiple phenotype expressions, and tripotential mesodermal capability. The authors report examples where electron microscopy (EM) plays a role in stem cell research. MSCs isolated from human arteries are ultrastructurally heterogeneous and become more homogenous after plastic adhesion. EM shows a moderate complement of organelles, mainly mitochondria, rough endoplasmic reticulum, and glycogen aggregates. Clear vacuoles and vesicles are prominent when cells are recovered from plates using an enzymatic method. Since the mesengenic plasticity is the single most important criterion to define a cell as mesenchymal stromal, the authors induced experimentally adipogenic, leiomyogenic, cardiomyogenic, osteo-chondrogenic differentiations. In no case did EM reveal the achievement of complete differentiation. The authors obtained multivacuolated pre-adipocytes and never univacuolated adipocytes typical of mature white fat; myofibroblast and rhabdomyoblast morphotypes, where contractile filaments were not organized to form functional complexes, i.e., dense bodies and sarcomeres. Chondrogenesis and osteogenesis assays resulted in extracellular matrix changes. Collagen and proteoglycan filament/particle deposition was seen when chondrogenesis was promoted. Hydroxyapatite crystals, psammoma bodies, and plaque-like calcified matrix deposits were found in the osteogenic matrix. EM provides detailed structural information on the degree of differentiation induced in stem cells and demonstrates that the methods so far developed are not able to promote complete cell differentiation. These observations contribute to explain why clinical applications with hMSCs have produced results far lower than initial expectations.

Myofibroblasts in interstitial lung diseases show diverse electron microscopic and invasive features.

The characteristic features of myofibroblasts in various lung disorders are poorly understood. We have evaluated the ultrastructure and invasive capacities of myofibroblasts cultured from small volumes of diagnostic bronchoalveolar lavage (BAL) fluid samples from patients with different types of lung diseases. Cells were cultured from samples of BAL fluid collected from 51 patients that had undergone bronchoscopy and BAL for diagnostic purposes. The cells were visualized by transmission electron microscopy and immunoelectron microscopy to achieve ultrastructural localization of alpha-smooth muscle actin (α-SMA) and fibronectin. The levels of α-SMA protein and mRNA and fibronectin mRNA were measured by western blot and quantitative real-time reverse transcriptase polymerase chain reaction. The invasive capacities of the cells were evaluated. The cultured cells were either fibroblasts or myofibroblasts. The structure of the fibronexus, and the amounts of intracellular actin, extracellular fibronectin and cell junctions of myofibroblasts varied in different diseases. In electron and immunoelectron microscopy, cells cultured from interstitial lung diseases (ILDs) expressed more actin filaments and α-SMA than normal lung. The invasive capacity of the cells obtained from patients with idiopathic pulmonary fibrosis was higher than that from patients with other type of ILDs. Cells expressing more actin filaments had a higher invasion capacity. It is concluded that electron and immunoelectron microscopic studies of myofibroblasts can reveal differential features in various diseases. An analysis of myofibroblasts cultured from diagnostic BAL fluid samples may represent a new kind of tool for diagnostics and research into lung diseases.

Albumin-induced epithelial mesenchymal transformation.

BACKGROUND: Progressive chronic kidney disease is often associated with albuminuria and renal fibrosis linked to the accumulation of myofibroblasts producing extracellular matrix. Renal myofibroblasts are derived from a number of cells including tubular epithelial cells (TECs) through epithelial mesenchymal transformation (EMT). This study explores the hypothesis that exposure of TECs to albumin induces EMT. METHODS: Normal rat TECs (NRK52E) were exposed in culture to de-lipidated bovine serum albumin (dBSA; 10 mg/ml) for 2, 4 and 6 days. Binding/uptake of fluoresceined albumin by PTCs was evaluated. Transformation into myofibroblasts was assessed by light and electron microscopy, immunofluorescence and Western blotting for α-smooth muscle actin (α-SMA), E-cadherin and transforming growth factor-ß1 (TGF-ß1). We also investigated the expression of fibroblast-specific protein-1 (FSP-1) and collagens I, III and IV. TGF-ß1 biological activity, mRNA and protein were measured. A neutralising anti-TGF-ß1 antibody was used to analyse the role of TGF-ß1 in albumin-induced EMT. RESULTS: Exposure of TECs to dBSA led to binding/uptake of albumin as well as fibroblastic morphological changes. Incubation of TECs with dBSA caused a reduction of TEC marker E-cadherin (ANOVA p = 0.0002) and de novo expression of fibroblast markers α-SMA and FSP-1 (ANOVA p = 0.0001) in a time-dependent manner. It also increased expression and activity of TGF-ß1. Neutralisation of TGF-ß1 significantly reduced EMT (p < 0.01). CONCLUSION: This study demonstrates that in vitro, albumin induces the transformation of TECs into cells with myofibroblast characteristics; a process that may be TGF-ß1 dependent.

Stromal cells in the human gut show ultrastructural features of fibroblasts and smooth muscle cells but not myofibroblasts.

The free spindled cells of the lamina propria of the gut have been reported as showing fibroblastic, smooth-muscle and myofibroblastic differentiation. A precise understanding of the differentiation of these cells is essential for appreciating their functions, and this paper addresses this question using ultrastructural analysis. Histologically normal samples from different areas of the gastrointestinal tract were studied. Both subepithelial stromal cells, lying immediately beneath the basal lamina, and the deeper interstitial stromal cells, were studied. Subepithelial and interstitial cells had comparable features, reinforcing the idea that these formed a single reticulum of cells. Two major cell types were identified. Some were smooth-muscle cells, on the basis of abundant myofilaments with focal densities, glycogen, an irregular cell surface, focal lamina and multiple attachment plaques alternating with plasmalemmal caveolae. Some cells had a lesser expression of these markers, especially of myofilaments, and were regarded as poorly differentiated smooth-muscle cells and descriptively referred to as 'myoid'. Other cells were fibroblastic to judge by prominent rough endoplasmic reticulum, an absence of myofilaments and lamina, but presence of focal adhesions. The fibronexus junctions of true myofibroblasts were not seen. The study emphasises that the smooth-muscle actin immunoreactivity in this anatomical site resides in smooth-muscle cells and not in myofibroblasts, a view consistent with earlier ultrastructural and immunostaining results. The recognition that these cells are showing smooth-muscle or fibroblastic but not true myofibroblastic differentiation should inform our understanding of the function of these cells.