Alpha-smooth Muscle Actin-positive Myofibroblasts Research Articles

TGF-β1 induces formation of TSG-6-enriched extracellular vesicles in fibroblasts which can prevent myofibroblast transformation by modulating Erk1/2 phosphorylation

Extracellular vesicles have emerged as important mediators of cell-to-cell communication in the pathophysiology of fibrotic diseases. One such disease is Peyronie’s disease (PD), a fibrotic disorder of the penis caused by uncontrolled transformation of resident fibroblasts to alpha-smooth muscle actin positive myofibroblasts. These cells produce large amounts of extracellular matrix, leading to formation of a plaque in the penile tunica albuginea (TA), causing pain, penile curvature, and erectile dysfunction. We have used primary fibroblasts derived from the TA of PD patients to explore the role of transforming growth factor beta 1 (TGF-β1), a key signalling factor in this process. TGF-β1 treatment elicited a range of responses from the myofibroblasts: (i) they secreted extracellular vesicles (EVs) that were more numerous and differed in size and shape from those secreted by fibroblasts, (ii) these EVs prevented TGF-β1-induced transformation of fibroblasts in a manner that was dependent on vesicle uptake and (iii) they prevented phosphorylation of Erk1/2, a critical component in modulating fibrogenic phenotypic responses, but did not affect TGF-β1-induced Smad-signalling. We posit that this effect could be linked to enrichment of TSG-6 in myofibroblast-derived EVs. The ability of myofibroblast-derived vesicles to prevent further myofibroblast transformation may establish them as part of an anti-fibrotic negative feedback loop, with potential to be exploited for future therapeutic approaches.

The Anti-Fibrotic Effect of Cold Atmospheric Plasma on Localized Scleroderma In Vitro and In Vivo.

Cold Atmospheric Plasma (CAP) has shown promising results in the treatment of various skin diseases. The therapeutic effect of CAP on localized scleroderma (LS), however, has not yet been evaluated. We investigated the effects of CAP on LS by comparing human normal fibroblasts (hNF), human TGF-β-activated fibroblasts (hAF), and human localized scleroderma-derived fibroblasts (hLSF) after direct CAP treatment, co-cultured with plasma-treated human epidermal keratinocytes (hEK) and with an experimental murine model of scleroderma. In hAF and hLSF, 2 min CAP treatment with the MicroPlaSterβ® plasma torch did not affect pro-fibrotic gene expression of alpha smooth muscle actin, fibroblast activating protein, and collagen type I, however, it promoted re-expression of matrix metalloproteinase 1. Functionally, CAP treatment reduced cell migration and stress fiber formation in hAF and hLSF. The relevance of CAP treatment was confirmed in an in vivo model of bleomycin-induced dermal fibrosis. In this model, CAP-treated mice showed significantly reduced dermal thickness and collagen deposition as well as a decrease in both alpha smooth muscle actin-positive myofibroblasts and CD68-positive macrophages in the affected skin in comparison to untreated fibrotic tissue. In conclusion, this study provides the first evidence for the successful use of CAP for treating LS and may be the basis for clinical trials including patients with LS.

Prognostic relevance of the loss of stromal CD34 positive fibroblasts in invasive lobular carcinoma of the breast

CD34+ fibroblasts are constitutive stromal components of virtually all organs, including the mammary stroma, being involved in matrix synthesis, antigen presentation, and tumor-associated stromal remodeling. The most common subtype of invasive breast carcinoma, invasive carcinoma of no special type (IBC-NST), is known for its stromal loss of CD34+ fibroblasts while acquiring alpha smooth muscle actin-positive (α-SMA+) myofibroblasts, i.e., cancer-associated fibroblasts (CAF), whereas invasive lobular carcinoma (ILC) displays partial preservation of CD34+ fibroblasts. The aim of this study was to evaluate the prognostic relevance of stromal CD34+ fibroblasts and α-SMA+ myofibroblasts in an extended collection of ILC. A total of 133 cases of ILC, primarily resected between 1996 and 2004 at University Hospital Marburg, were examined semiquantitatively for stromal content of CD34+ fibroblasts and α-SMA+ myofibroblasts. Partial preservation of CD34+ fibroblasts in the tumor stroma of ILC was confirmed. Absence of CD34+ fibroblasts in the tumor stroma significantly correlated with the presence of α-SMA+ myofibroblasts (p = 0.010), positive lymph node status (p = 0.004), and pN stage (p = 0.006). Stromal loss of CD34+ fibroblasts was significantly associated with lower overall and disease-free survival rates (p = 0.012 and 0.013, respectively). Multivariate analysis adjusted for pT and pN stage revealed stromal loss of CD34+ fibroblasts as independent prognostic parameter (p = 0.05). To our knowledge, this is the first report defining prognostically relevant stromal subtypes of ILC with long-term follow-up. Future research targeting the potential diagnostic and therapeutic implications of CD34+ fibroblasts and CAF in breast cancer, especially ILC, is a promising field of interest.

Consistency of color-deconvolution for analysis of image intensity of alpha smooth muscle actin-positive myofibroblasts in solid multicystic ameloblastomas

ABSTRACT Ameloblastoma is an odontogenic tumor with a slow, locally aggressive growth pattern and multiple clinico-histologic types. The number of stromal myofibroblasts within ameloblastoma often is correlated with growth and aggressiveness. Color-deconvolution to separate different colors of immunostained tissues is a promising approach to quantifying myofibroblasts in tumors such as ameloblastoma. We investigated the reliability of the color-deconvolution method using cross-sectional design to evaluate alpha-smooth muscle actin (α-SMA)-positive myofibroblasts in solid multicystic ameloblastoma. Formalin fixed tissues of eight cases of solid multicystic ameloblastoma were immunostained for α-SMA using the horseradish peroxidase-diaminobenzidine (HRP-DAB) method. Color-deconvolution using ImageJ software was used to quantify the staining intensity of brown DAB α-SMA stained myofibroblasts. Color-deconvoluted images of brown DAB stained tissues exhibited distinct morphological features of solid multicystic ameloblastoma with α-SMA stained myofibroblasts distributed abundantly adjacent to the ameloblastoma epithelial islands. The computed image intensity of α-SMA stained myofibroblasts was quantitatively similar among the different ameloblastoma samples. A combination of color-deconvolution and α-SMA staining of myofibroblasts is a useful diagnostic tool for evaluating histologic differentiation and growth pattern of ameloblastoma.

Idiopathic Myocardial Fibrosis in Captive Chimpanzees (Pan troglodytes).

Cardiovascular disorders and predominantly idiopathic myocardial fibrosis are frequently associated with mortality among zoo-housed chimpanzees (Pan troglodytes). Formalin-fixed whole hearts of deceased chimpanzees housed in zoos (n = 33) and an African sanctuary (n = 2) underwent detailed macroscopic and histopathologic examination using a standardized protocol. Archived histological slides from the hearts of 23 additional African sanctuary-housed chimpanzees were also examined. Myocardial fibrosis (MF) was identified in 30 of 33 (91%) of the zoo-housed chimpanzees but none of the 25 sanctuary-housed chimpanzees. MF was shown to be characterized by both interstitial and replacement fibrosis. Immunophenotyping demonstrated that the fibrotic lesions were accompanied by the increased presence of macrophages, alpha smooth muscle actin-positive myofibroblasts, and a minimal to mild T-cell-dominant leukocyte infiltration. There was no convincing evidence of cardiotropic viral infection or suggestion that diabetes mellitus or vitamin E or selenium deficiency were associated with the presence of the lesion. However, serum vitamin D concentrations among zoo-housed chimpanzees were found to be lower in seasons of low ultraviolet light levels.

Activated Fibroblast Program Orchestrates Tumor Initiation and Progression; Molecular Mechanisms and the Associated Therapeutic Strategies.

Neoplastic epithelial cells coexist in carcinomas with various non-neoplastic stromal cells, together creating the tumor microenvironment. There is a growing interest in the cross-talk between tumor cells and stromal fibroblasts referred to as carcinoma-associated fibroblasts (CAFs), which are frequently present in human carcinomas. CAF populations extracted from different human carcinomas have been shown to possess the ability to influence the hallmarks of cancer. Indeed, several mechanisms underlying CAF-promoted tumorigenesis are elucidated. Activated fibroblasts in CAFs are characterized as alpha-smooth muscle actin-positive myofibroblasts and actin-negative fibroblasts, both of which are competent to support tumor growth and progression. There are, however, heterogeneous CAF populations presumably due to the diverse sources of their progenitors in the tumor-associated stroma. Thus, molecular markers allowing identification of bona fide CAF populations with tumor-promoting traits remain under investigation. CAFs and myofibroblasts in wound healing and fibrosis share biological properties and support epithelial cell growth, not only by remodeling the extracellular matrix, but also by producing numerous growth factors and inflammatory cytokines. Notably, accumulating evidence strongly suggests that anti-fibrosis agents suppress tumor development and progression. In this review, we highlight important tumor-promoting roles of CAFs based on their analogies with wound-derived myofibroblasts and discuss the potential therapeutic strategy targeting CAFs.

A Step Forward Toward the Understanding of the Long-Term Pathogenesis of Double Capsule Formation in Macrotextured Implants: A Prospective Histological Analysis.

Although increasingly reported in the literature, most plastic surgeons cannot define the etiology of double capsules. Often an incidental finding at implant exchange, double capsules are frequently associated with macrotextured devices. Several mechanisms have been proposed, including at the forefront that shearing causes a delamination of the periprosthetic capsule into a double capsule. This study was designed to confirm the hypothesis that mechanical forces are involved in formation of double capsules by histological analysis. A prospective analysis of consecutive implants with double capsules removed over 2 years was performed. Data collected at the time of surgery included Baker classification, reason for explant, implant manufacturer and style, and any presence of a seroma associated with the capsule. Specimens were sent for analysis by histology utilizing hematoxylin and eosin and alpha-smooth muscle actin staining techniques. Eight double capsules were collected for specimen analysis. All capsules demonstrated evidence of granulation tissue, alpha-smooth muscle actin positive myofibroblasts, and folds with embedded texture. Fibrosis surrounded weak areas with presence of layering and splitting, creating a potential space that is prone to separation. Tears and folds from granulomatous reaction are also present within the outer layer of the double capsule, which can only be explained by a mechanical shearing force as a pathogenic mechanism. Understanding the pathogenesis of double capsules may allow plastic surgeons to refine their indications for macrotextured implants while providing guidance to patients on avoidance of activities that produce shear-forces. The findings support the hypothesis that shearing forces delaminate the capsule into 2 separate distinct capsules.

Abstract PR11: Hematopoietic system aging has profound effects on the breast tumor microenvironment and cancer progression

Abstract The majority of breast neoplasms are diagnosed in patients who are between the ages of 65 and 80. Although tumor growth is generally less aggressive and metastasis is less prevalent in older patients than in younger patients, elderly patients suffer the highest mortality and morbidity associated with breast cancer and breast cancer treatment. The reasons for age-dependent differences in incidence and aggressiveness of breast cancer are not understood. Treatment of breast cancer patients therefore presents age-specific challenges. We recently established mouse and human xenograft breast tumor models that mimic the age dependent disease progression that is observed in breast cancer patients, whereby tumor cells implanted into aged mice had lower incidence and slower growth kinetics than when implanted into young mice. Moreover, alpha-smooth muscle actin-positive myofibroblasts within the tumor microenvironment were significantly less prevalent in the tumors from aged mice than in size-matched tumors from young mice. We discovered that bone marrow derived cells from aged mice were significantly less effective in supporting breast cancer progression than the counterpart cells from young mice. Strikingly, bone marrow derived cells from young mice rescued tumor growth and stromal desmoplasia in aged mice. We are elucidating some of the molecular and phenotypic differences between young and aged tumor-promoting bone marrow cells. For example, we found that bone marrow cells from aged mice express lower levels of the cytokine, granulin, which is known to activate normal mammary fibroblasts in young mice. Our findings indicate that the age of the host hematopoietic system is a powerful determinant of breast tumor progression. Defining functional and molecular differences between tumor supportive hematopoietic cells in young and aged hosts should lay a foundation for further work in this relatively uncharted area of cancer research. Understanding tumor support mechanisms that exist in young and aged hosts should suggest new ideas for preclinical development of age-stratified breast cancer therapies. Citation Format: Timothy Marsh, Irene Wong, Amey Barakat, Yuanbo Qin, Sandra S. McAllister. Hematopoietic system aging has profound effects on the breast tumor microenvironment and cancer progression. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr PR11. doi:10.1158/1538-7445.CHTME14-PR11

Interleukin-6-dependent phenotypic modulation of cardiac fibroblasts after acute myocardial infarction.

Interleukin-6 (IL-6) is a multifunctional cytokine that orchestrates the immune response to a wide variety of pathophysiologic challenges but also contributes to tissue homeostasis. Furthermore, IL-6 is elevated in patients with acute myocardial infarction. Hyaluronan (HA) is an extracellular carbohydrate that has been implicated in wound healing and accumulates after acute myocardial infarction (AMI). Aim of this study was to investigate the involvement of IL-6 in the regulation of the HA-matrix in the early phase of infarct healing. In the present study, we show by the use of a blocking anti-IL-6 antibody, that endogenous IL-6 rapidly but transiently increased HA-synthase (HAS) 1 and 2 expression resulting in the formation of a HA-rich matrix acutely after AMI in mice. In vitro, IL-6 induced HAS1 and 2 via STAT3 phosphorylation in cardiac fibroblasts (CF) and supported a myofibroblastic phenotype in a HA-dependent manner. Furthermore, CCL5 and MCP1 expression were dependent on IL-6, HA-synthesis and the HA-receptor CD44 as shown in cultured CF derived from CD44 knockout mice. In vivo after AMI, blocking IL-6 decreased HA-matrix formation in the peri-infarct region and alpha-smooth muscle actin-positive myofibroblasts. Blocking IL-6 also reduced neutrophil infiltration in infarcted left ventricles. Moreover, treatment with the blocking IL-6 antibody reduced cardiac ejection fraction and increased infarct size 3weeks after AMI. These findings support a functionally important role for IL-6 in CF by transiently inducing a HA-rich matrix that in turn promotes a myofibroblastic phenotype and inflammatory responses, and ultimately establishes a cardioprotective program after AMI.

Small mammary analogue secretory carcinoma arising from minor salivary gland of buccal mucosa

Mammary analogue secretory carcinoma (MASC) of salivary gland has been identified at 2010 by Skalova et al. There are only four cases occurring in buccal mucosa among the previously reported 70 cases. In this article, we present such fifth case. A 59-year-old Japanese man noticed the nodule measuring 5 mm in maximum diameter of the left buccal mucosa and subsequent biopsy showed the finding of tubular and microcystic configurations of the tumor cells. Secretory materials in glandular lumens were positive for periodic acid–Schiff stain with and without diastase treatment. Immunohistochemically, neoplastic cells demonstrated diffuse immunolabeling for vimentin, cytokeratin 7, cytokeratin 19 and S-100 protein. Additionally, the bilayer structure of CD34-positive stromal cells and alpha-smooth muscle actin-positive myofibroblasts in the capsule was identified. Finally, otolaryngologists and oral surgeon should bear in mind that MASC may occasionally arise from buccal mucosa. CD34-positive stromal cells and myofibroblasts may be associated with the regulation of tumor growth of MASC, like other salivary gland tumors. This is the smallest tumor among the previously reported cases.

Deterioration of fracture healing in the mouse model of NF1 long bone dysplasia

Neurofibromatosis type 1 (NF1) is an autosomal dominant genetic disease resulting from inactivating mutations in the gene encoding the protein neurofibromin. NF1 manifests as a heritable susceptibility to tumours of neural tissue mainly located in the skin (neurofibromas) and pigmented skin lesions. Besides these more common clinical manifestations, many NF1 patients (50%) have abnormalities of the skeleton. Long bones are often affected (usually the tibia) and the clinical signs range from bowing to spontaneous fractures and non-unions. Here we present the analysis of bone fracture healing in the Nf1Prx1-knock-out mouse, a model of NF1 long bone dysplasia. In line with previously reported cortical bone injury results, fracture healing was impaired in Nf1Prx1 mice. We showed that the defective fracture healing in Nf1Prx1 mice is characterized by diminished cartilaginous callus formation and a thickening of the periosteal bone. These changes are paralleled by fibrous tissue accumulation within the fracture site. We identify a population of fibrous tissue cells within the Nf1 deficient fracture as alpha-smooth muscle actin positive myofibroblasts. Additionally, histological and in-situ hybridization analysis reveal a direct contact of the fracture site with muscle fascia, suggesting a possible involvement of muscle derived cells in the fracture deterioration.

Wound Contraction Is Significantly Reduced by the Use of Microcarriers to Deliver Keratinocytes and Fibroblasts in anIn VivoPig Model of Wound Repair and Regeneration

In full-thickness injuries caused by extensive burns or penetrating traumatic injuries, the natural epidermal stem cell niche is destroyed, and wound healing occurs through migration of cells from the wound edges and wound contraction. This can lead to significant contracture formation, especially in large full-thickness injuries, causing lack of mobility and pain. Contraction is reduced when wounds are treated using split-thickness skin grafts (STSG) or dermal substitutes, particularly in combination with cultured autologous keratinocytes, delivered as confluent sheets or sprayed as a single cell suspension (SAK). Here, we show that the application of keratinocytes alone or keratinocytes with fibroblasts, delivered on microcarriers, in combination with STSG or a dermal substitute, significantly reduces contraction of wounds in vivo in a porcine model of wound repair and regeneration. A decrease in alpha-smooth muscle actin-positive myofibroblasts, the cell type responsible for wound contraction, accompanies the reduction in contraction. These findings demonstrate the potential for a significant clinical advantage in the treatment of full-thickness injuries.

Abstract B22: Oncogene activation of K-ras in resident myofibroblasts leads to development of spontaneous bronchioalveolar carcinoma in mice

Abstract Stromal myofibroblasts, also known as carcinoma-associated fibroblasts, are recruited to neoplastic lesions and act as accomplices in promoting tumor progression. By secreting various growth factors, cytokines, extracellular matrix molecules, and matrix remodeling proteins, stromal myofibroblasts enhance tumor cell proliferation, angiogenesis, and tumor cell invasion. In contrast, the role of resident myofibroblasts in tumorigenesis is largely unknown. Our preliminary results strongly suggest that resident myofibroblasts may act as contributors to tumorigenesis. Selective expression of the oncogenic mutant of K-ras (K-rasG12D) in alpha smooth muscle actin-positive (αSMA+) stromal myofibroblasts is achieved by crossing αSMA-Cre+ transgenic mice to the LSL-KrasG12D f/+ mouse model (hereafter αSMAKrasG12D). While αSMAKrasG12D mice were phenotypically normal at birth, these mice developed spontaneous bronchioalveolar carcinoma by 5 weeks of age at 100% tumor penetrance. We speculate that genetic mutations in stromal myofibroblasts can disrupt stromal-epithelial homeostasis, driving hyperproliferation of epithelial cells. This fosters further accumulation of genetic mutations in the epithelial compartment, and thus, result in carcinogenesis. Citation Information: Cancer Res 2009;69(23 Suppl):B22.

Histologic Analysis of Acellular Dermal Matrix in the Treatment of Anal Fistula in an Animal Model

Human acellular dermal matrix (ADM) has been used successfully for the treatment of severe burns, ureter support, and abdominal wall reconstruction. This study was designed to evaluate the mechanism of ADM in the closure of anal fistula in an experimental porcine model. The fistula-in-ano model was created in the porcine model and treated with ADM in 14 animals. Fistula specimens were obtained at hours 12 and 24 and on days 3, 7, 14, 28, 60. Hematoxylin and eosin staining, Masson trichrome staining, and immunohistochemical staining for alpha smooth muscle actin and matrix metalloproteinase 9 were performed. The cell density increased from hour 12 to day 7 and decreased from day 7 to day 28 (p < 0.001). Mature vessels stained with alpha smooth muscle actin were identified at day 7. Alpha smooth muscle actin-positive myofibroblasts were found in clusters at the edge of the ADM at day 7. The density of vessels (p < 0.001) and myofibroblasts (p < 0.001) increased from day 7 to day 14. The density of matrix metalloproteinase 9 increased from hour 12 to day 7 and decreased from day 14 to day 60 (p < 0.001). Partially organized bundles of muscle were found by day 60. We suggest that ADM is a reasonable new option for closure of anal fistulas. Anal fistulas begin to heal as early as 12 hours, and day 7 may be an important time point to judge whether the fistula healed preliminarily or not. The ability of ADM to become vascularized and remodeled by autologous cells may be advantageous for anal fistula healing.

PET and Macro- and Microautoradiographic Studies Combined with Immunohistochemistry for Monitoring Rat Intestinal Ulceration and Healing Processes

18F-FDG PET is used mainly in clinical settings for imaging focal cancer sites, but the usefulness of the modality in imaging gastrointestinal ulcers has not been established. We investigated whether PET can be used for noninvasive monitoring of indomethacin-induced small-intestine ulceration. Intestinal ulcers were induced in rats by subcutaneous administration of indomethacin. An 18F-FDG PET scan was obtained at 1, 2, and 7 d after indomethacin administration. 18F-FDG uptake in the small intestine was quantified by gamma-counting, and macro- and microautoradiographic studies were performed to determine the site of 18F-FDG uptake in tissue and at the cellular level. Ulcers observed in the intestine (mainly in the ileum) 1-4 d after indomethacin administration were most severe at 1 d after administration and were almost healed at day 7. The PET study showed increased 18F-FDG uptake in the intestine correlating to the severity of ulceration, returning to the basal level on day 7. Ex vivo imaging and gamma-counting showed that these regions of high uptake corresponded to regions of ulceration. A microautoradiographic study combined with immunohistochemistry revealed heavy accumulation of 18F-FDG in inflammatory cells containing peroxidase on day 1 and in cells forming granulation tissue (alpha-smooth muscle actin-positive myofibroblasts and ED2-positive macrophages) on days 2-4 in and around ulcers. Proliferating (Ki67-immunopositive) intestinal crypt cells were also densely labeled with 18F-FDG in intact intestinal tissue taken from the indomethacin-treated and the control animals. Our experimental data suggest that 18F-FDG PET may be useful for evaluating the occurrence of small-intestine ulcers. Ulceration could be visualized early by the prominent uptake of 18F-FDG by inflammatory cells and by the formation of granulation tissue by cells in and around ulcers.

Stromal Fibroblasts Activated by Tumor Cells Promote Angiogenesis in Mouse Gastric Cancer

Myofibroblasts, also known as activated fibroblasts, constitute an important niche for tumor development through the promotion of angiogenesis. However, the mechanism of stromal fibroblast activation in tumor tissues has not been fully understood. A gastric cancer mouse model (Gan mice) was recently constructed by simultaneous activation of prostaglandin (PG) E2 and Wnt signaling in the gastric mucosa. Because both the PGE2 and Wnt pathways play a role in human gastric tumorigenesis, the Gan mouse model therefore recapitulates the molecular etiology of human gastric cancer. Microvessel density increased significantly in Gan mouse tumors. Moreover, the expression of vascular endothelial growth factor A (VEGFA) was predominantly induced in the stromal cells of gastric tumors. Immunohistochemistry suggested that VEGFA-expressing cells in the stroma were alpha-smooth muscle actin-positive myofibroblasts. Bone marrow transplantation experiments indicated that a subset of gastric myofibroblasts is derived from bone marrow. Importantly, the alpha-smooth muscle actin index in cultured fibroblasts increased significantly when stimulated with the conditioned medium of Gan mouse tumor cells, indicating that gastric tumor cells activate stromal fibroblasts. Furthermore, conditioned medium of Gan mouse tumor cells induced VEGFA expression both in embryonic and gastric fibroblasts, which further accelerated the tube formation of human umbilical vein endothelial cells in vitro. Notably, stimulation of fibroblasts with PGE2 and/or Wnt1 did not induce VEGFA expression, thus suggesting that factors secondarily induced by PGE2 and Wnt signaling in the tumor cells are responsible for activation of stromal fibroblasts. Such tumor cell-derived factors may therefore be an effective target for chemoprevention against gastric cancer.

Differential cutaneous wound healing in thermally injured MRL/MPJ mice

Adult wound repair occurs with an initial inflammatory response, reepithelialization, and the formation of a permanent scar. MRL/MpJ mice following ear-hole punch biopsies display accelerated healing and tissue regeneration. In this study, we characterized the healing responses in both MRL/MpJ and BALB/c mice following a 15% total body surface area full-thickness cutaneous burn injury. Macroscopic and histological observations show that delayed wound closure in MRL/MpJ mice is accompanied by an increase in edema, reduced neutrophil infiltration, and more prominent eschar. In vivo bromodeoxyuridine labeling showed no defect in keratinocyte proliferation and migration (reepithelialization). In comparison with BALB/c mice, MRL/MpJ wounds had greater collagen deposition, less granulation tissue formation, and contained fewer alpha-smooth muscle actin-positive myofibroblasts. An observed reduction in dermal neutrophil infiltration and myofibroblast development correlated with enhanced angiogenesis. Overall, BALB/c wounds contracted sooner and to a larger degree, resulting in a significant decrease in scar formation. Interestingly, MRL/MpJ mice showed overt abnormalities in hair follicle proliferation, morphogenesis, and subsequent hair regrowth postburn injury. No substantial evidence of tissue regeneration was observed in either BALB/c or MRL/MpJ wounds. Our results convincingly demonstrate that MRL/MpJ skin burn wounds heal with scar formation with delays in two critical wound healing events: wound closure, and myofibroblast development.

Angiotensin Receptor-1 Blocker Inhibits Atherosclerotic Changes and Endothelial Disruption of the Aortic Valve in Hypercholesterolemic Rabbits

We sought to examine the effect of angiotensin receptor blocker (ARB) on the formation of lesions in the aortic valves of hypercholesterolemic rabbits. Recently, atherosclerosis has been recognized as a mechanism that is responsible for calcific aortic stenosis. The effect of ARBs might help to prevent aortic stenosis because they have multiple antiatherosclerotic effects. Male Japanese white rabbits (n = 36) were separated as follows: control with chow diet (C) and vehicle (V) groups, both of which were fed a 1% cholesterol diet for 8 weeks, and an ARB group (A), which was fed a 1% cholesterol diet for 8 weeks with ARB (olmesartan, 1 mg/kg/day) for the last 4 weeks. This dose of olmesartan did not affect either blood pressure or cholesterol levels. Dietary cholesterol induced fatty deposition with macrophage accumulation and osteopontin coexpression in valve leaflets, whereas ARB decreased macrophage accumulation (% area: V, 9.3 +/- 0.34; A, 1.4 +/- 0.30; p = 0.003) and osteopontin expression (p = 0.017). Angiotensin-converting enzyme was also up-regulated in V and decreased by olmesartan (p = 0.015). Immunohistochemistry with anti-CD31 antibody revealed that dietary cholesterol disrupted and olmesartan preserved endothelial integrity on the lesion-prone aortic side of the valve (% CD31-positive circumference: V, 30 +/- 3.7; A, 62 +/- 4.8; p = 0.003). Numbers of alpha-smooth muscle actin-positive myofibroblasts were increased in V and decreased by olmesartan (p = 0.003). Real-time polymerase chain reaction revealed that increased amounts of messenger ribonucleic acid for osteoblast-specific transcription factor core binding factor alpha-1 in V were diminished by olmesartan. Atherosclerotic changes in the aortic valves of rabbits fed with cholesterol were inhibited by ARB, whereas endo-thelial integrity was preserved and transdifferentiation into myofibroblasts and/or osteoblasts in valve leaflets was inhibited.

Bone Marrow–Derived Myofibroblasts Contribute to the Renal Interstitial Myofibroblast Population and Produce Procollagen I after Ischemia/Reperfusion in Rats

Bone marrow-derived cells (BMDC) have been proposed to exert beneficial effects after renal ischemia/reperfusion injury (IRI) by engraftment in the tubular epithelium. However, BMDC can give rise to myofibroblasts and may contribute to fibrosis. BMDC contribution to the renal interstitial myofibroblast population in relation to fibrotic changes after IRI in rats was investigated. A model of unilateral renal IRI (45 min of ischemia) was used in F344 rats that were reconstituted with R26-human placental alkaline phosphatase transgenic BM to quantify BMDC contribution to the renal interstitial myofibroblast population over time. After IRI, transient increases in collagen III transcription and interstitial protein deposition were observed, peaking on days 7 and 28, respectively. Interstitial infiltrates of BMDC and myofibroblasts reached a maximum on day 7 and gradually decreased afterward. Over time, an average of 32% of all interstitial alpha-smooth muscle actin-positive myofibroblasts coexpressed R26-human placental alkaline phosphatase and, therefore, were derived from the BM. BMD myofibroblasts produced procollagen I protein and therefore were functional. The postischemic kidney environment was profibrotic, as demonstrated by increased transcription of TGF-beta and decreased transcription of bone morphogenic protein-7. TGF-beta protein was present predominantly in interstitial myofibroblasts but not in BMD myofibroblasts. In conclusion, functional BMD myofibroblasts infiltrate in the postischemic renal interstitium and are involved in extracellular matrix production.

Effects of Gadolinium Chloride (GdCl 3) on the Appearance of Macrophage Populations and Fibrogenesis in Thioacetamide-Induced Rat Hepatic Lesions

Macrophages infiltrating injured tissue play an important part in fibrogenesis. To shed light on the functional roles of macrophages, we investigated the appearance of macrophage populations in thioacetamide (TAA)-induced rat hepatic lesions, with or without pretreatment with GdCl(3), a chemical capable of inhibiting Kupffer cell functions. In the GdCl(3)+TAA group rats received a single intraperitoneal injection of GdCl(3) (7.5mg/kg body weight) and, after 24h, a single intravenous injection of TAA (300mg/kg body weight). Rats in the TAA group received TAA only. Rats in both groups were examined on post-TAA injection (PTI) days 3, 5, and 7. In the TAA group, on PTI day 3, when TAA-induced hepatocyte injury was particularly prominent, the number of macrophages peaked, subsequently decreasing until PTI day 7. As compared with the TAA group, the GdCl(3)+TAA group showed significantly decreased numbers of ED1-immunolabelled cells (exudate macrophages) and ED2-immunolabelled cells (Kupffer cells) on PTI days 3, 5, and 7, and OX6-immunolabelled cells (antigen-presenting macrophages) on PTI days 3 and 5. Although less strikingly, the numbers of alpha-smooth muscle actin-positive myofibroblasts and fibrotic areas were decreased in the GdCl(3)+TAA group. By RT-PCR, the expression of TGF-beta1 mRNA was suppressed on PTI days 3 and 7 in the GdCl(3)+TAA group, and the suppressed expression was confirmed in vitro by treating rat macrophage-like cells (HS-P) with 1% GdCl(3). The study showed that GdCl(3) treatment decreased the numbers of macrophages appearing in hepatic lesions and inhibited TGF-beta1 mRNA expression in macrophages. Decreased numbers of macrophages may contribute to improvement of hepatic fibrosis.