α-SMA Positive Cells Research Articles

Adenoid cystic carcinoma arising the anterior lingual gland (Blandin–Nuhn gland)

The present case describes a rare example of adenoid cystic carcinoma (ACC) arising the Blandin-Nuhn gland. A 75-year-old Japanese woman took a partial glossectomy, hemimandiblectomy, excision of the hypoglossal nerve and a skin grafting. The tumor tissue consisted of tubular type of ACC and perineural invasion. The tumor cells were immunoreactive for S-100, keratin, α-smooth muscle actin (α-SMA) and PCNA. Numbers of PCNA and α-SMA positive cells were higher in modified or perineural tumor cells than that in tubular tumor cells. Biological roles of ACC invasion might be related to exist of PCNA and actin.

Fascin-positive dendritic cells and fibroblastic reticulum cells build a framework of T-cell areas in lymph nodes.

Fascin, a 55-kDa actin-bundling protein, and alpha-smooth muscle actin (ASMA) were immunohistochemically examined in murine normal and stimulated lymph nodes. In specific pathogen-free young female mice, a few fascin-positive cells (FPCs) were located in the sinus and surrounding tissues, but ASMA-positive cells were undetectable. Following a subcutaneous injection of sheep red blood cells, the numbers of FPCs and their dendrites increased in the paracortex, with the accumulation of activated lymphocytes. Fibroblastic reticulum cells (FRCs), endothelial cells, histiocytic cells and lymphocytes in various stages of maturation were all fascin negative. These results indicated that fascin could be a reliable marker of paracortical dendritic cells in murine lymph nodes. However, FRCs became ASMA positive. Immunoelectron microscopy showed that the FPCs were interdigitating cells and that they closely contacted with FRCs. These two types of cells and reticular fiber formed a network in the paracortex and contacted with each other. In active paracortical response, both FPCs and FRCs are also stimulated and might play a significant role in the maturation of the lymphocytes.

Effect of nitric oxide on the differentiation of fibroblasts into myofibroblasts in the Peyronie’s fibrotic plaque and in its rat model

The myofibroblast shares phenotypic features of both fibroblasts and smooth muscle cells. It plays a critical role in collagen deposition and wound healing and disappears by apoptosis when the wound is closed. Its abnormal persistence leads to hypertrophic scar formation and other fibrotic conditions. Myofibroblasts are present in the fibrotic plaque of the tunica albuginea (TA) of the penis in men with Peyronie’s disease (PD), a localized fibrosis that is accompanied by a spontaneous induction of the inducible nitric oxide synthase (iNOS), also observed in the TGFβ1-elicited, PD-like lesion in the rat model. iNOS expression counteracts fibrosis, by producing nitric oxide (NO) that reduces collagen deposition in part by neutralization of profibrotic reactive oxygen species. In this study we investigated whether fibroblast differentiation into myofibroblasts is enhanced in the human and rat PD-like plaque and in cultures of human tissue fibroblasts. We also examined whether NO reduces this cell differentiation and collagen synthesis. The myofibroblast content in the fibroblast population was measured by quantitative immunohistochemistry as the ratio between α-smooth muscle actin (ASMA; myofibroblast marker) and vimentin (general fibroblast marker) levels. We found that myofibroblast content was considerably increased in the human and TGFβ1-induced rat plaques as compared to control TA. Inhibition of iNOS activity by chronic administration of l-iminoethyl- l-lysine to rats with TGFβ1-induced TA lesion increased myofibroblast abundance and collagen I synthesis measured in plaque and TA homogenates from animals injected with a collagen I promoter construct driving the expression of β-galactosidase. Fibroblast differentiation into myofibroblasts occurred with passage in the cell cultures from the human PD plaque, but was minimal in cultures from the TA. Induction of iNOS in PD and TA cultures with a cytokine cocktail and a NO donor, S-nitroso- N-acetyl penicillamine (SNAP), was detected by immunohistochemistry. Both treatments reduced the total number of cells and the number of ASMA positive cells, whereas only SNAP decreased collagen I immunostaining. These results support the hypotheses that myofibroblasts play a role in the development of the PD plaque and that the antifibrotic effects of NO may be mediated at least in part by the reduction of myofibroblast abundance and lead to a reduction in collagen I synthesis.

Tissue inhibitor of metalloproteinases-1 attenuates spontaneous liver fibrosis resolution in the transgenic mouse

It has been suggested that the tissue inhibitor of metalloproteinases-1 (TIMP-1) is involved in spontaneous resolution of liver fibrosis. The aim of this study was to investigate whether TIMP-1 altered spontaneous resolution of liver fibrosis in conjunction with matrix metalloproteinases (MMP) inhibition and hepatic stellate cell (HSC) activation. The livers of liver-targeted TIMP-1 transgenic (TIMP-Tg) and control hybrid (Cont) mice were harvested at 0, 3, 7, and 28 days following spontaneous recovery from CCl 4-induced liver fibrosis. The extent of fibrosis resolution, MMP expression, α-smooth-muscle actin (α-SMA) positive cells, and procollagen-(I) messenger RNA (mRNA) in the liver were assessed at the respective periods in both groups. We also examined the effect of TIMP-1 on HSC apoptosis. The TIMP-Tg mice showed significantly attenuated resolution of spontaneous liver fibrosis compared with the Cont mice. The hydroxyproline content, number of α-SMA positive cells, and procollagen-(I) mRNA rapidly decreased with time in the Cont mice, whereas these markers were little changed in TIMP-Tg mice. The level of the active form of metalloproteinases-2 (MMP-2) in the TIMP-Tg mice was less than that in the Cont mice. TIMP-1 markedly decreased the nonparenchyma apoptotic cells in the liver fibrosis resolution model, and it also inhibited HSC apoptosis associated with suppression of caspase-3 activity in vitro. In conclusion, TIMP-1 significantly attenuated spontaneous resolution of liver fibrosis by the combination of a net reduction of the MMP activity and suppression of apoptosis in HSC. (H EPATOLOGY 2002;36:850-860.)

Expression of Nuclear Transcription Factor PAX2 in Renal Biopsies of Juvenile Nephronophthisis

PAX2, a homeotic gene of ‘paired box family’, is a nuclear transcription factor expressed in mesenchymal/epithelial conversion during the early stages of nephrogenesis; however, its repression is necessary for terminal differentiation of mature tubular cells. Transgenic overexpression in animal model causes epithelial hyperproliferation and microcyst formation. In humans, PAX2 expression has been observed in cystic and dysplasic tubular epithelia in kidney malformation and in kidney disease. We have investigated PAX2 expression and its colocalization with cytokeratin and/or vimentin in 17 biopsies of juvenile nephronophthisis (NPH), an autosomal-recessive renal disease characterized by diffuse renal fibrosis and occasional cysts. Fourteen cases were analyzed for deletion and mutation in the NPH1 gene locus and 33% resulted to be deleted or mutated; for the remaining cases the diagnosis was based on clinical and pathological criteria. The control group included 4 congenital dysplastic kidneys, and 10 biopsies of nephropathies with secondary chronic tubulointerstitial damage. In all cases of renal dysplasia a strong nuclear positivity was observed in immature tubules surrounded by αSMA-positive mesenchymal cells. In NI biopsies the tubular epithelia were almost PAX2 negative, although tubulointerstitial damage was severe. In 14/17 NPH1 moderate-to-strong nuclear PAX2 positivity of tubular cells was observed, particularly in cystic distal tubules located at the corticomedullary junction, but also in proximal tubular sections. The PAX2 signal co-localized more with cytokeratin staining than with vimentin. Our results confirm the observation of PAX2 expression in immature dysplastic tubules and its repression in mature renal tubular cells, also in the presence of severe secondary interstitial fibrosis. PAX2 seems to be overexpressed in NPH. The genetic defect of NPH, a disease probably due to a primary defect along the cascade of mesenchymal epithelial differentiation, could generate a functionally abnormal protein involved in focal adhesion signaling and cell/matrix interaction. The failure of PAX2 repression or its reactivation in NPH could be a marker of hyperproliferation and incomplete maturation of epithelial tubular cells, probably due to a defect cell/matrix cross-talk, and involved in interstitial fibrosis and cysts formation.

Participation of α-smooth muscle actin-positive cells in renomedullary interstitial cell tumors

Renomedullary interstitial cell tumors are benign lesions which are generally discovered in specimens nephrectomized for other malignant tumors or by autopsy. We examined the histologic features of eight tumors from four patients and investigated the appearance of alpha-smooth muscle actin (ASMA)-positive cells in these tumors using immunohistochemistry. We considered that five tumors are cellular type and the remaining three as fibrous. Characteristic hyalinization was observed in two of the three fibrous tumors. All the tumors except for one fibrous type contained entrapped tubular cells. CD35-positive cells (dendritic cells) and ASMA-positive cells were observed in all the tumors, with a more frequent occurrence in the cellular type than the fibrous type. CD35-negative spindle cells were considered as fibroblasts or activated fibroblasts (myofibroblasts). The number of CD35-positive cells was higher than that of ASMA-positive cells. Additionally, the entrapped tubular cells showed the transition to spindle cells and some of them expressed for ASMA. With double immunohistochemical staining, there were some cells showing positive reactions for both CD35 and ASMA. Furthermore, an ultrastructural examination confirmed the presence of ASMA-positive filaments in the dendritic cells and myofibroblasts. The expression of TGF-beta 1 was observed not only in the tumor cells and the collecting ducts surrounding the tumor but also in the entrapped tubular cells. In addition, the intensity of TGF-beta 1 was stronger in/around the tumor than in the areas distant from the tumor. The positive cells were more numerous in the cellular type than in the fibrous type. In conclusion, ASMA-positive cells appear in renomedullary interstitial cell tumors and some of the cells may originate in dendritic interstitial cells, fibroblasts including myofibroblasts, and entrapped tubular cells. Furthermore, TGF-beta 1 may contribute to the formation of fibrosis in the tumors.

Angiotensin-II type 1 receptor interaction is a major regulator for liver fibrosis development in rats

The renin-angiotensin system (RAS) is frequently activated in patients with chronic liver diseases. Angiotensin-II (AT-II) has been suggested to play an important role in liver fibrogenesis. It induces hepatic stellate cell (HSC) proliferation and up-regulates the transforming growth factor β1 (TGF-β1) expression via AT-II type 1 receptor (AT1-R) in vitro. The aim of the present study was to examine the in vivo effect of candesartan (CA), a clinically used AT1-R blocker (ARB), and perindopril (PE), an angiotensin-converting enzyme (ACE) inhibitor (ACE-I), on pig serum-induced liver fibrosis development in rats. The clinically available comparable doses of CA and PE significantly attenuated the fibrosis development. These inhibitory effects of PE and CA were also found in the on-going liver fibrosis model. The hepatic hydroxyproline and serum fibrosis markers were significantly suppressed by CA and PE treatment. Furthermore, the α smooth muscle actin (α-SMA) positive cells in number were markedly suppressed by CA and PE treatment. Similarly, the hepatic TGF-β1 protein and messenger RNA (mRNA) levels were significantly suppressed. Our in vitro study showed that AT-II increased the TGF-β1 mRNA expression in the activated HSCs, and this effect was totally blocked by CA. These results suggested that the RAS, especially AT-II and AT1-R interaction plays a pivotal role in liver fibrosis development through HSC activation. Because both CA and PE are widely used in clinical practice without serious side effects, these drugs may provide an effective new strategy for anti–liver fibrosis therapy.

Alpha-smooth muscle actin expression upregulates fibroblast contractile activity.

To evaluate whether alpha-smooth muscle actin (alpha-SMA) plays a role in fibroblast contractility, we first compared the contractile activity of rat subcutaneous fibroblasts (SCFs), expressing low levels of alpha-SMA, with that of lung fibroblasts (LFs), expressing high levels of alpha-SMA, with the use of silicone substrates of different stiffness degrees. On medium stiffness substrates the percentage of cells producing wrinkles was similar to that of alpha-SMA-positive cells in each fibroblast population. On high stiffness substrates, wrinkle production was limited to a subpopulation of LFs very positive for alpha-SMA. In a second approach, we measured the isotonic contraction of SCF- and LF-populated attached collagen lattices. SCFs exhibited 41% diameter reduction compared with 63% by LFs. TGFbeta1 increased alpha-SMA expression and lattice contraction by SCFs to the levels of LFs; TGFbeta-antagonizing agents reduced alpha-SMA expression and lattice contraction by LFs to the level of SCFs. Finally, 3T3 fibroblasts transiently or permanently transfected with alpha-SMA cDNA exhibited a significantly higher lattice contraction compared with wild-type 3T3 fibroblasts or to fibroblasts transfected with alpha-cardiac and beta- or gamma-cytoplasmic actin. This took place in the absence of any change in smooth muscle or nonmuscle myosin heavy-chain expression. Our results indicate that an increased alpha-SMA expression is sufficient to enhance fibroblast contractile activity.

Pancreatic ductal myofibroblasts. Proliferative patterns in various pathologic situations.

Myofibroblasts in the periacinar area of the pancreas have been demonstrated to mediate fibrogenesis in pancreatic fibrosis. However, only a few reports have described myofibroblasts in the pancreatic duct. To elucidate the presence of myofibroblasts in the pancreatic ductal wall, we performed an immunohistochemical study, using immunostains for both alpha-smooth muscle actin (alphaSMA) and desmin, and an electron microscopic study on surgically resected pancreatic specimens from 10, 23, 23, and 56 cases of focal pancreatitis (FP), chronic pancreatitis (CP), pancreatic carcinoma (PCa), and carcinoma of the papilla of Vater (VPCa), respectively. All cases showed localized stenosis of the main pancreatic duct by means of preoperative pancreatography. As controls, 20 autopsy cases were studied. alphaSMA-positive and desmin-negative cells existed in the ductal walls of controls and were revealed as myofibroblasts by means of electron microscopy. In six FPs, proliferation of myofibroblasts was observed at the stenotic portion. In VPCas, myofibroblasts mainly proliferated in the pancreatic ductal wall. In CPs and PCas, no myofibroblast proliferation was observed at the stenotic portion. The proliferation of myofibroblasts might occur as a wound healing process in FP, while acting against elevation of intraductal pressure in VPCa. In conclusion, proliferation of myofibroblasts plays an important role in ductal changes in various pathological situations.

Tissue inhibitor of metalloproteinases-1 promotes liver fibrosis development in a transgenic mouse model

Tissue inhibitor of metalloproteinases-1 (TIMP-1) has been shown to be increased in liver fibrosis development both in murine experimental models and human samples. However, the direct role of TIMP-1 during liver fibrosis development has not been defined. To address this issue, we developed transgenic mice overexpressing human TIMP-1 (hTIMP-1) in the liver under control of the albumin promoter/enhancer. A model of CCl 4-induced hepatic fibrosis was used to assess the extent of fibrosis development in TIMP-1 transgenic (TIMP-Tg) mice and control hybrid (Cont) mice. Without any treatment, overexpression of TIMP-1 itself did not induce liver fibrosis. There were no significant differences of pro-(α1)-collagen-I, (α2)-collagen-IV, and α-smooth muscle actin (α-SMA) mRNA expression in the liver between TIMP-Tg and Cont-mice, suggesting that overexpression of TIMP-1 itself did not cause hepatic stellate cell (HSC) activation. After 4-week treatment with CCl 4, however, densitometric analysis revealed that TIMP-Tg-mice had a seven-fold increase in liver fibrosis compared with the Cont-mice. The hepatic hydroxyproline content and serum hyaluronic acid were also significantly increased in TIMP-Tg-mice, whereas CCl 4-induced liver dysfunction was not altered. An active form of matrix metalloproteinases-2 (MMP-2) level in the liver of TIMP-Tg-mice was decreased relative to that in Cont-mice because of the transgenic TIMP-1. Immunohistochemical analysis revealed that collagen-I and collagen-IV accumulation was markedly increased in the liver of CCl 4-treated TIMP-Tg-mice with a pattern similar to that of α-SMA positive cells. These results suggest that TIMP-1 does not by itself result in liver fibrosis, but strongly promotes liver fibrosis development. (Hepatology2000;32:1248-1254.)

Myofibroblast Differentiation in the Connective Tissues of the Amnion and Chorion of Term Human Fetal Membranes—Implications for Fetal Membrane Rupture and Labour

An area of the fetal membranes, within the rupture tear after spontaneous delivery at term, exhibits altered morphology compared to more distal sites. It is characterized by marked swelling of the amniotic and chorionic connective tissue layers, consistent with structural weakness, and a marked reduction of the thickness of both the cytotrophoblast and decidual layers. These features, albeit less extreme, have been identified in fetal membranes in the lower uterine pole in patients prior to labour. In this study of pre-labour, labour-affected and post-labour term fetal membranes, we report that these regions are associated with an alteration in the phenotype of the vimentin positive mesenchymal cell population of the chorionic connective tissue reticular layer, and are consistent with myofibroblastic differentiation, i.e. α-smooth muscle actin (α-sma) expression. In the reticular layer of the lower uterine pole biopsies in the labour-affected group the numbers and densities of α-sma immunoreactive positive cells were 17-fold (P=0.04) and 8.5-fold (P=0.02) higher than in mid-zone biopsies. After delivery, in rupture line biopsies the numbers and densities were 50-fold (P=0.002) and 36-fold (P=0.003) higher compared to mid zone biopsies. The percentage of the vimentin positive population positive for α-sma was 2–5 per cent in mid-zone biopsies compared to 49 per cent (P=0.03) in the labour-affected ‘cervical’ biopsies and 69 per cent (P=0.05) in the rupture line biopsies. Within the tear sites, α-sma positive cells were also detected within the fibroblastic layer of the amniotic connective tissue. Although there was no significant difference between the numbers and density of α-sma cells in the reticular layers between mid and lower uterine pole biopsies in the pre-labour group, in a proportion of patients the biopsies were similar to labour-affected biopsies indicating that this alteration occurs prior to clinically apparent labour in these patients. The incidence of α-sma positive cells in the reticular layer correlated with morphological changes within the fetal membranes, for example thickness of reticular (r2=0.349, P=0.0006) and amniotic connective tissue layers (r2=0.389, P=0.0002). This suggests that cellular activities associated with myofibroblastic differentiation in the reticular layer of the chorion may be associated with the observed connective tissue changes, fetal membrane rupture and labour.

The expression of G1 cyclins and RB-E2F and the effect of vitamin E on hepatic stellate cells activated by acute CCL4 induced hepatic injury

K.S. Lee, K.J. Lee, K.H. Ham C.Y. Chon, Y.M. Moon Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Korea. Hepatic stellate cells(HSC) play a key role in hepatic fibrogenesis. Therefore HSC activation model is needed to find some agents to block HSC activation. We evaluated HSC activation and proliferation, G1-S cyclins expression, Rb-E2F expression and the effect of vitamin E during acute CCh induced hepatic injury. Methods: Sprague-Dawtey rats(100g) each received a single intraperitoneal injection(IP) of CCh in mineral oil at a dose of 2ml/kg body weight(Group I and II). In addition, animals received IP of 200mg/kg vitamin E(Group II) daily, 2 days before CCI4 administration until the sacrifice. 5 animals(each time, each group) were sacrificed at Oh, 8h, 16h, 32h, 48h and 60h after CCh injection and 30pCi of [~H]Thymidine was administered 4h before sacrifice. Blood samples were obtained to check serum ALT level. Liver were promptly removed and a piece was fixed and embedded in paraffin for aSMA immunohistochemical(IMH) staining and count aSMA positive cells in 400x HPF. HSC were isolated from fresh fiver by non0erfusion method and single step density Nycodenz gradient. Nuclei were prepared for gel shift assay(E2F). DNA(10#g) were prepared from HSC to check [~H]Thymidine uptake by using B counter. Protein(10pg: Rb, CDK2, CDK4 / 2Ozg: cyclin D1, cyclin E) were prepared from HSC for western blot. Results: The serum ALT level changed 74-+20.7, 170-+54.3, 258+ 83.5, 1178-+381.3, 274-+174.0, 92-+74.0 in group I and 64-+21.9, 152-+58.9, 156-+97.6, 576-+141.5, 70-+35.4, 62+55.4 in group II(peaked at 32h: P<0.01 / decrease in group I]: P<0.06). [°H]Thymidine uptake(c0m) changed 76.4 +-14.7, 76.6-+19.7, 78.8-+23.8, 529.2-+284.8, 299.0-+161.6, 179.6-+63.9 in g_+roup I and 71.6-+19.9, 90.4-+9.6, 85.0-+2A.0, Z23.0-+86.3, 171.2-+47.8, 127.8 19.3 in group II(oeaked at 32h: P<0.01 / decrease in group I/: P<0.05). IMI-I for aSMA showed 0, 0, 2.0-+0.8, 49.2-+6.8, 31.2-+13.6, 21.8-+10.1 in group I and 0, 0, 0, 14.8-+1.4, 18.1-+9.1, 12.2-+6.2 in group N(peaked at 32h: P<0.01 / decrease in group 1]: P<0.01). In western blot, cyclin D1 and Rb showed bands at 32h and cyclin E, CDK2 and CDK4 showed bands at 16h, 32h, 48h. All bands were diminished in group II. In gel shift assay, E2F showed shifted bands at 16h, 32h, 48h and NFkB showed shifted band at 32h. All shifted bands were diminished in group II. Conclusion: The serum ALT level, HSC proliferation and activation were peaked at 32h after CCI4 administration and significantly decreased in vitamin E treated group. G1-S cyclins, CDK2, CDK4, Rb showed bands at 1611, 32h, 48h or 32h after CC14 administration and diminished in vitamin E treated group. E2F showed shifted bands at 16h, 32h, 4811 and NFkB showed shifted band at 32h. All shifted bands were diminished in vitamin E treated group.

Vascular and Interstitial Changes in the Peritoneum of Capd Patients with Peritoneal Sclerosis

To analyze morphological changes in the peritoneum of peritoneal sclerosis (PS) patients. Emphasis was put on vascular abnormalities, because the continuous exposure to glucose-based dialysis solutions could cause diabetiform changes and because longitudinal transport studies suggested the development of a large peritoneal vascular surface area. Peritoneal biopsies from continuous ambulatory peritoneal dialysis (CAPD) patients were investigated in two studies. Diabetic patients were excluded. In study 1, 11 PS biopsies were compared to three control groups varying in duration of CAPD treatment: 0 months (n = 15), 2 - 25 months (n = 7), and > 25 months CAPD (n = 7). The second study was a case-control study, comparing six biopsies from the long-term control group to six PS biopsies, matched for age and duration of CAPD. All biopsies were scored for presence and type of fibrosis [Picro Sirius red, type IV collagen, alpha-smooth muscle actin (alphaSMA)] and for neoangiogenesis (factor VIII). Thickening of vascular walls by type IV collagen and vasodilation of capillaries were measured by computer-aided planimetry. In study 1 the presence of sclerosing fibrosis, deposition of interstitial type IV collagen, and the number of myofibroblasts (alphaSMA-positive cells) was greater in the PS biopsies than biopsies from all control groups (p < 0.002). Moreover, the number of vessels per field was higher in PS biopsies (p < 0.01). Vascular wall thickening of small arteries (p < 0.008) and vasodilation of capillaries were found in PS biopsies compared to all control groups (p < 0.007). The second study revealed differences in the presence of sclerosis but not in the extent of fibrosis between PS biopsies and their controls. The number of vessels per field in PS biopsies was higher compared to controls (p = 0.04). Also, thickening of the vascular wall was more marked in PS biopsies (p = 0.03). Vasodilation of capillaries was greater in PS biopsies than in controls (p = 0.07). Fibrosis of the peritoneum may precede peritoneal sclerosis. The deposition of type IV collagen and the presence of myofibroblasts in the interstitial layer could be part of a pathologic process similar to the scarring in diabetic nephropathy. Neoangiogenesis and thickening of the vascular wall by type IV collagen are consistent with glucose-induced microangiopathy.These abnormalities and the vasodilation of the capillaries can explain the high dialysate-to-plasma ratios or mass transfer area coefficients of low molecular weight solutes that can be found in long-term CAPD patients.

Quantitative Analysis of the Interstitial Myofibroblasts in Idiopathic Mesangiocapillary Glomerulonephritis Type I

Fourteen renal biopsy specimens from patients with mesangiocapillary glomerulonephritis type I (MCGN-I), for whom both light and electron microscopies as well as immunofluorescence microscopy and full clinical data were available, were examined quantitatively. As a control, 10 biopsy specimens of the kidney from patients with minimal change disease (MCD) were used. Morphometric investigations were performed by a computer image analysis system to investigate whether myofibroblasts have a role in tubulointerstitial fibrosis in MCGN-I and, in particular, to examine the relationship between alpha-SMA expression and interstitial infiltrates. The morphometric study revealed that in MCGN-I patients the mean values for the expression of alpha-SMA, interstitial volume, CD68+, CD45RB+, CD43+ and CD20+ cells were significantly increased, as compared with a control group. In the MCGN-I group, there were significant positive correlations between the interstitial expression of alpha-SMA and the interstitial volume as well as CD68+ and CD45RB+ cells. The correlations between the interstitial expression of alpha-SMA and CD43+ as well as CD20+ cells were positive, but without statistical significance. In conclusion, our study suggests that interstitial a-SMA positive cells play a role in the development of interstitial fibrosis in MCGN-I. The significant correlation between the interstitial expression of alpha-SMA and interstitial CD68+ cells may identify monocytes/macrophages as important mediators in the process of inducing the myofibroblast phenotype in resting fibroblasts.

Expression of Cytoskeletal Proteins Differentiates between Progressors and Non-Progressors in Treated Idiopathic Membranous Nephropathy

Myofibroblasts play an important role in wound healing in a variety of tissue injuries. They have also been implicated in tissue fibrosis including renal scarring. This study was aimed at defining their role in one of the commonest forms of nephrotic syndrome in adults, namely membranous nephropathy. We have studied 21 patients with biopsy proven idiopathic membranous nephropathy who were treated with glucocorticoids, attempting to define the role of myofibroblasts (α-smooth muscle actin-positive as well as vimentin-positive cells) in the progression of this form of nephropathy. There were 13 non-progressors (NP) and 8 progressors (P). The clinical, histological, and immunohistochemical characteristics of both groups were compared. Immunohistochemical staining for myofibroblasts cytoplasmic markers α-smooth muscle actin (α-SMA) and vimentin relied on an avidin-biotin-peroxidase method. The level of blood pressure, degree of proteinuria, severity of interstitial infiltrate and interstitial fibrosis did not differentiate P from NP. However, vascular sclerosis was more severe in P compared to NP (p < 0.016) and its severity predicted the subsequent functional outcome (slope of the 1/serum creatinine against time; r² = 0.618, p < 0.01). Mesangial α-SMA was significantly higher in P (31 ± 18.6%) than in NP (14.5 ± 9.8%), p < 0.015. Interstitial α-SMA immunostain was also higher in P but did not reach statistical significance. However, the number of interstitial myofibroblasts (α-SMA positive cells) closely predicted the subsequent rate of the progression of chronic renal failure (r² = 0.919, p < 0.0001). Mesangial vimentin expression was not different between both groups. By contrast, interstitial vimentin immunostain was higher in P (19.1 ± 8.8%) compared to NP (7.9 ± 5.6%), p < 0.002. These data suggest that the expression of mesangial and interstitial cytoskeletal proteins (α-SMA and vimentin) may have useful prognostic implications as they appear to differentiate between patients with membranous nephropathy who respond to immunosuppression and those who continue to progress.

Demonstration of collagen type VI and alpha-smooth muscle actin in renal fibrotic injury in man.

Overproduction of collagenous fibres types I and III is a common finding of fibrotic injury. Collagen type VI is generally associated with type I. Appearance of fibroblasts expressing alpha-smooth muscle actin (ASMA) and their role in fibrogenesis has been partly defined. However, correlation between renal fibroblasts and accumulation of microfibrillar collagen type VI, as well as its exact distribution, is not fully delineated. This study was undertaken to investigate these issues using a complex morphological approach. Morphological examination included immunohistochemical detection of the collagen type VI and ASMA, relying on a streptavidin-biotin-peroxidase-based technique, and electron microscopy. Collagen type VI was strongly expressed in areas of fibrotic injury, although mild expression was always revealed in renal interstitium. Glomerular immunoreactivity with the anti-collagen type VI antibody was almost nil excepting cases of diabetic glomerulosclerosis and amyloid nephrosis. Glomerular nodules in cases of diabetes displayed intense reactivity. Mesangial, as well as discontinuous peripheral deposition of collagen along the glomerular basement membrane, was noticed in case of amyloidosis. Ultrastructurally, cross-banded collagen microfibrils were found in renal interstitium in close association with the fibroblast membrane. Moreover, fibrillar elements revealing tubular structure and fine filamentous material were observed between cross-banded microfibrils. Some of fibroblasts exhibited bundles of microfilaments in their cytoplasm. An increased number of ASMA-positive cells was detected in fibrotic interstitium. An intense concentric network made up of actin-bearing cells surrounded glomerular capillaries in the case of crescentic glomerular lesions. Markedly increased deposition of collagen type VI takes place in renal fibrotic lesions. Simultaneously, interstitial fibrotic areas appeared to contain a great number of fibroblasts sharing morphological characteristics of classic fibroblasts and smooth muscle cells. Detailed examination of coexistence of these two interstitial phenomena should further clarify the cellular mechanisms involved in renal interstitial fibrosis.

Prolyl 4-hydroxylase inhibitor (HOE 077) inhibits pig serum-induced rat liver fibrosis by preventing stellate cell activation

Background/Aims: The aim of this study was to investigate the effect and mechanism of fibrosuppression by a newly synthesized prolyl 4-hydroxylase inhibitor {HOE 077, 2, 4-pyridine dicarboxylic acids bis [(2-methoxyethyl) amide]} on pig serum-induced liver fibrosis in the rat.Methods: Male Wistar rats received 0.5 ml of pig serum twice a week for 10 weeks with 0, 100 or 200 ppm of HOE 077. At the end of the experiment, the hydroxyproline content of the liver, and alanine aminostransferase were measured. Histological stains used were HE, azan and a stain for α-smooth muscle actin (α-SMA). Electron microscopy was also performed. Messenger RNA expressions of type I and III procollagen were examined by Northern blot analysis. α-SMA positive cells and fibers with azan staining were assessed as percent area of the tissue specimen, using an image analysis system.Results: Rats that received pig serum for 10 weeks showed an increased liver hydroxyproline content of 318±39 μg/g we weight (n=15). HOE 077 at doses up to 200 ppm significantly (p<0.01) reduced this increase of liver hydroxyproline content (181±39 μg/g wet weight, n=15) in accordance with imporved histological findings. 200 ppm of HOE 077 significantly reduced mRNA expressions of α2(I) (486±102 vs 151±36, p<0.01) and α1(III) (276±127 vs 160±67, p<0.05) procollagen and percent area of α-SMA positive cells (2.94±2.14 vs 1.17±0.88%). Electron microscopy revealed that 200 ppm of HOE 077 prevented the loss of fat droplets.Conclusions: A prolyl 4-hydroxylase inhibitor (HOE 077) prevented pig serum-induced rat liver fibrosis by inhibiting stellate cell activation.

Evidence that “myofibroblast-like” cells are the cellular source of capsular collage in hepatocellular carcinoma

Background/Aims: The prognosis for patients with hepatocellular carcinoma is poor although tumour encapsulation has been associated with improved survival and disease-free rates. While the source of the tumour capsule is unclear, the major role that activated hepatic stellate cells play in the deposition of liver matrix in normal and diseased states suggests the possible involvement of these cells in tumour encapsulation. Methods: Twenty-four liver tumours (seven encapsulated HCC, seven non-encapsulated HCC, 10 colorectal metastases) were studied. Activated hepatic stellate cells were identified by immunohistochemistry for α-smooth muscle actin (α-SMA) and in situ hybridization for pro-collagen α 1 (I) mRNA. Collagen deposition was localized using Masson's trichrome strain. Results: Pro-collagen α 1 (I) mRNA co-localized to α-SMA positive hepatic stellate cells within the region of increased collagen deposition in (i) the tumour capsule of encapsulated HCC, and (ii) the tumour junction of non-encapsulated HCC and colorectal metastis. In addition, there was marked peritumour expression of α-SMA and procollagen α 1 (I) mRNA, which diminished with distance away from the tumour in all tumour groups. The degree of expression was greatest with encapsulated HCC, less with non-encapsulated HCC and least with colorectal metastasis. This contrasted with the absence of α-SMA expression in normal liver from the same patients. Within the tumours, colorectal metastases differed from HCC by demonstrating marked α-SMA expression and collagen deposition in the septa. Conclusions: Our findings demonstrate that activated hepatic stellate cells (i) are responsible for increased peritumour collagen production in non-encapsulated HCC and colorectal metastasis, and (ii) may be implicated in tumour capsule formation in HCC and metastasis stroma development. Thus, stellate cells may influence the local hepatic invasion by these tumours.

Alpha-smooth muscle actin-positive stromal cells in cholangiocarcinomas, hepatocellular carcinomas and metastatic liver carcinomas

Aims/Methods: In the human liver, α-smooth muscle actin (ASMA) is present in smooth muscle of the vasculature, perisinusoidal cell (Ito cells), and myofibroblasts derived from perisinusoidal cells. In this study, we investigated ASMA-positive stromal cells and their relation to tumor fibrosis in 50 cholangiocarcinomas, 30 hepatocellular carcinomas, and 57 metastatic liver carcinomas. Results: Tumor fibrosis was much more extensive in cholangiocarcinomas and metastatic liver carcinomas than in metastatic liver carcinomas. ASMA immunoreactivity was prominent in the sinusoids surrounding cancer nodules and in the cancerous stroma, not in sinusoids remote from cancer nodules. ASMA-positive stromal cells were divisible into peritumoral ASMA-positive perisinusoidal cells and intratumoral ASMA-positive stromal cells. Both types of ASMA-positive cells were abundant in cholangiocarcinomas and metastatic liver carcinomas, but much more scanty in hepatocellular carcinomas. The number of both types showed a significant positive correlation with the degree of tumor fibrosis. The peritumoral ASMA-positive perisinusoidal cells were frequently in direct continuity with intratumoral ASMA-positive stromal cells in cholangiocarcinomas and metastatic liver carcinomas. Conclusions: These findings show that ASMA-positive stromal cells are related to tumor fibrosis in liver malignancies. Although direct evidence is lacking, the data suggest that, in cholangiocarcinomas and metastatic liver carcinomas, peritumoral ASMA-positive perisinusoidal cells transform into activated perisinusoidal cells (myofibroblasts), are incorporated into the tumor (intratumoral ASMA-positive stromal cells), and produce extracellular matrix proteins, that lead to tumor fibrosis. The scantly ASMA-positive cells in hepatocellular carcinomas may in part be responsible for the small amount of fibrosis in this tumor.

Spatial Distribution of Connexin43, the Major Cardiac Gap Junction Protein, Visualizes the Cellular Network for Impulse Propagation From Sinoatrial Node to Atrium

Myocytes are electrically coupled by gap junctions, which are composed of low-resistance intercellular channels. The major cardiac gap junction protein is connexin43 (Cx43). The distribution of Cx43 has been studied by immunofluorescence to visualize the electrical coupling between atrial tissue and sinoatrial node. From modeling studies, this coupling was inferred to be gradual in order to shield the sinoatrial node from the atrial hyperpolarizing influence. The actual Cx43 labeling pattern did not show the expected gradient but instead a rather black and white staining in a striking pattern of strands of cells. We used an immunohistochemical marker (anti-alpha-smooth muscle actin [alpha SMA]) that specifically cross-reacts with guinea pig sinoatrial node cells together with Cx43 antibody to stain previously electrophysiologically mapped sinoatrial nodes. We found that in the guinea pig sinoatrial node the impulse originates in an alpha SMA-positive, virtually Cx43-negative, region (primary pacemaker region). The impulse then travels obliquely upward to the crista terminalis through a region where layers of alpha SMA-positive cells alternate with layers of Cx43-positive SMA-negative cells. The layers of Cx43-positive cells appear to become broader and thicker in the direction of the crista terminalis, whereas the layers of alpha SMA-positive cells become thinner and narrower. Lateral contacts between Cx43- and alpha SMA-positive cells were very sparse and only detected where the Cx43-positive strands ended (the region where alpha SMA-positive cells fill the whole space between endocardium and epicardium, ie, the putative primary pacemaker region). From these results, we conclude that the primary pacemaker is shielded from the hyperpolarizing influence of the atrium by a gradient in coupling brought about by tissue geometric factors rather than by a gradient of gap junction density.