Cells Expressing Α-smooth Muscle Actin Research Articles

Differential Biological Effects of Dietary Lipids and Irradiation on the Aorta, Aortic Valve, and the Mitral Valve.

AimsDietary cholesterol and palmitic acid are risk factors for cardiovascular diseases (CVDs) affecting the arteries and the heart valves. The ionizing radiation that is frequently used as an anticancer treatment promotes CVD. The specific pathophysiology of these distinct disease manifestations is poorly understood. We, therefore, studied the biological effects of these dietary lipids and their cardiac irradiation on the arteries and the heart valves in the rabbit models of CVD.Methods and ResultsCholesterol-enriched diet led to the thickening of the aortic wall and the aortic valve leaflets, immune cell infiltration in the aorta, mitral and aortic valves, as well as aortic valve calcification. Numerous cells expressing α-smooth muscle actin were detected in both the mitral and aortic valves. Lard-enriched diet induced massive aorta and aortic valve calcification, with no detectable immune cell infiltration. The addition of cardiac irradiation to the cholesterol diet yielded more calcification and more immune cell infiltrates in the atheroma and the aortic valve than cholesterol alone. RNA sequencing (RNAseq) analyses of aorta and heart valves revealed that a cholesterol-enriched diet mainly triggered inflammation-related biological processes in the aorta, aortic and mitral valves, which was further enhanced by cardiac irradiation. Lard-enriched diet rather affected calcification- and muscle-related processes in the aorta and aortic valve, respectively. Neutrophil count and systemic levels of platelet factor 4 and ent-8-iso-15(S)-PGF2α were identified as early biomarkers of cholesterol-induced tissue alterations, while cardiac irradiation resulted in elevated levels of circulating nucleosomes.ConclusionDietary cholesterol, palmitic acid, and cardiac irradiation combined with a cholesterol-rich diet led to the development of distinct vascular and valvular lesions and changes in the circulating biomarkers. Hence, our study highlights unprecedented specificities related to common risk factors that underlie CVD.

Cytoglobin-expressing cells in the splenic cords contribute to splenic fibrosis in cirrhotic patients.

Among several noninvasive evaluation methods of portal hypertension (PH), the measurement of spleen stiffness is a reliable method for predicting esophageal variceal bleeding; however, the underlying mechanisms for increased stiffness remain unclear. We attempted to elucidate the pathological changes to the spleen and the underlying mechanisms in patients with PH. Histological examination was performed using splenic tissues from 42 patients with PH who underwent laparoscopic splenectomy, and the results were compared with those from patients without PH. In addition to splenic sinus congestion, diffuse fibrosis was detected in the splenic cords in the red pulp of patients with PH. The degree of the fibrosis was well correlated with severity in thrombocytopenia and splenomegaly. Cells expressing α-smooth muscle actin dramatically increased in the splenic cord. Cytoglobin (Cygb) expression was detected in human splenic cords as reported in animal reticular cells, and fluorescent double immunostaining revealed that these cells expressed α-smooth muscle actin in patients with PH, suggesting transformation of Cygb-expressing cells to myofibroblastic cells. Expression levels of nicotinamide adenine dinucleotide phosphate oxidase (NOX) 2, nitrotyrosine, and transforming growth factor-β were markedly upregulated in the red pulp of patients with PH, implying a significant role of oxidative stress in the mechanism for splenic fibrosis. Splenic fibrosis progresses along with advancement of PH. Cygb-expressing cells in the splenic cord possibly participate in this process through mechanisms including oxidative stress.

Mouse Sertoli cells isolation by lineage tracing and sorting.

Sertoli cells play a key role in spermatogenesis by supporting the germ cells throughout differentiation. The isolation of Sertoli cells is essential to study their functions. However, the close contact of Sertoli cells with other testicular cell types and the high proliferation of contaminating cells are obstacles to obtain pure primary cultures. Current rodent Sertoli cell isolation protocols result in enriched, rather than pure Sertoli cells. Therefore, novel approaches are necessary to improve the purity of Sertoli cell primary cultures. The goal of this study is to obtain pure mouse Sertoli cells using lineage tracing and fluorescence-activated cell sorting (FACS). We bred the Amh-Cre mouse line with tdTomato line to generate mice constitutively expressing red fluorescence specifically in Sertoli cells. Primary cultures of Sertoli cells isolated from prepubertal mice showed that 79% of cells expressed tdTomato, as evaluated by fluorescence microscopy and flow cytometry; however, nearly all adherent cells were positive for vimentin. Most of the tomato-negative cells expressed α-smooth muscle actin (α-SMA), a peritubular myoid cell marker, but double-negative populations were also present. These findings suggest that vimentin lacks Sertoli cell-specificity and that α-SMA is not adequate to identify all of the contaminating cells. Upon FACS sorting; however, virtually 100% of the cells were tdTomato positive, expressed vimentin, but not α-SMA. Prepubertal mice yielded a higher number of Sertoli cells compared to adults, but both could be adequately sorted. In conclusion, our study shows that lineage tracing and sorting is an efficient strategy for acquiring pure populations of murine Sertoli cells.

Translational relevance of the goat as a preclinical model of the human labrum and chondrolabral junction-histological study.

The purpose of this study was to evaluate the histologic features of the caprine labrum, with emphasis on the chondrolabral junction, with the goal of informing the feasibility of the goat as an animal model. The left hip joint of six adolescent Spanish goats (Capra pyrenaica) was harvested and subjected to anatomical and histological assessments. Human acetabular and femoral head samples, collected during total hip arthroplasty, served as comparison samples. The caprine labrum was found to consist of mostly type I collagen with uniform crimp, with an average crimp length of 20.8 µm. Upon histological assessment, acetabular articular chondrocytes were found to express substance-P, especially near or in the chondrolabral junction. And the majority of nonvascular cells expressed α-smooth muscle actin (SMA), with no notable elastin and laminin expression. Human labrum demonstrated similar staining patterns. Overall, the goat hip was found to be homologous to the human hip, demonstrating potential as a useful animal model for future studies. This is the first report of a crimped collagen structure in the labrum. Crimped type I collagen at the chondrolabral junction imparts an extension-recovery property which allows for toleration of stress without permanent deformation, underlying the importance of its preservation during surgery. The high expression of substance-P reflects the degree to which the labrum is innervated. Finally, the expression of α-SMA with contractile characteristics could indicate the potential for chondrocyte (i.e., myochondrocytes) modeling of the extracellular matrix. Statement of Clinical Significance: Establishment of a large animal model and deeper knowledge of the histological composition of the hip joint will enhance our study of the acetabular labrum, including repair techniques. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:1070-1080, 2020.

Impact of HIV/simian immunodeficiency virus infection and viral proteins on adipose tissue fibrosis and adipogenesis

HIV-infected patients receiving antiretroviral treatment (ART) often present adipose tissue accumulation and/or redistribution. adipose tissue has been shown to be an HIV/SIV reservoir and viral proteins as Tat or Nef can be released by infected immune cells and exert a bystander effect on adipocytes or precursors. Our aim was to demonstrate that SIV/HIV infection per se could alter adipose tissue structure and/or function. Morphological and functional alterations of subcutaneous (SCAT) and visceral adipose tissue (VAT) were studied in SIV-infected macaques and HIV-infected ART-controlled patients. To analyze the effect of Tat or Nef, we used human adipose stem cells (ASCs) issued from healthy donors, and analyzed adipogenesis and extracellular matrix component production using two dimensional (2D) and three-dimensional (3D) culture models. Adipocyte size and index of fibrosis were determined on Sirius red-stained adipose tissue samples. Proliferating and adipocyte 2D-differentiating or 3D-differentiating ASCs were treated chronically with Tat or Nef. mRNA, protein expression and secretion were examined by RT-PCR, western-blot and ELISA. SCAT and VAT from SIV-infected macaques displayed small adipocytes, decreased adipogenesis and severe fibrosis with collagen deposition. SCAT and VAT from HIV-infected ART-controlled patients presented similar alterations. In vitro, Tat and/or Nef induced a profibrotic phenotype in undifferentiated ASCs and altered adipogenesis and collagen production in adipocyte-differentiating ASCs. We demonstrate here a specific role for HIV/SIV infection per se on adipose tissue fibrosis and adipogenesis, probably through the release of viral proteins, which could be involved in adipose tissue dysfunction contributing to cardiometabolic alterations of HIV-infected individuals.

Bone morphogenetic protein-7 inhibits endothelial-mesenchymal transition in pulmonary artery endothelial cell under hypoxia.

Pulmonary artery hypertension (PAH) is characterized by structural changes in pulmonary arteries. Increased numbers of cells expressing α-smooth muscle actin (α-SMA) is a nearly universal finding in the remodeled artery. It has been confirmed endothelial-to-mesenchymal transition (EndoMT) may be a source of those α-SMA-expressing cells. In addition, the EndoMT is reversible. Here, we show that under hypoxia, the expression of bone morphogenetic protein 7 (BMP-7) was decreased both in vivo and in vitro. We also found that under normoxia, BMP-7 deficiency induced spontaneous EndoMT and cell migration. The hypoxia-induced EndoMT and cell migration were markedly attenuated after pretreatment with rh-BMP-7. Moreover, m-TOR phosphorylation was involved in EndoMT and BMP-7 suppressed hypoxia-induced m-TORC1 phosphorylation in pulmonary artery endothelial cells. Our results demonstrate that BMP-7 attenuates the hypoxia-induced EndoMT and cell migration by suppressing the m-TORC1 signaling pathway. Our study revealed a novel mechanism underlying the hypoxia-induced EndoMT in pulmonary artery endothelial cells and suggested a new therapeutic strategy targeting EndoMT for the treatment of pulmonary arterial hypertension.

Effect of Methotrexate on an In Vitro Patient-Derived Model of Proliferative Vitreoretinopathy.

PurposeThe purpose of this study was to develop a method for isolating, culturing, and characterizing cells from patient-derived membranes in proliferative vitreoretinopathy (PVR) to be used for drug testing.MethodsPVR membranes were obtained from six patients with grade C PVR. Membrane fragments were analyzed by gross evaluation, fixed for immunohistologic studies to establish cell identity, or digested with collagenase II to obtain single cell suspensions for culture. PVR-derived primary cultures were used to examine the effects of methotrexate (MTX) on proliferation, migration, and cell death.ResultsGross analysis of PVR membranes showed presence of pigmented cells, indicative of retinal pigment epithelial cells. Immunohistochemistry identified cells expressing α-smooth muscle actin, glial fibrillary acidic protein, Bestrophin-1, and F4/80, suggesting the presence of multiple cell types in PVR. Robust PVR primary cultures (C-PVR) were successfully obtained from human membranes, and these cells retained the expression of cell identity markers in culture. C-PVR cultures formed membranes and band-like structures in culture reminiscent of the human condition. MTX significantly reduced the proliferation and band formation of C-PVR, whereas it had no significant effect on cell migration. MTX also induced regulated cell death within C-PVR as assessed by increased expression of caspase-3/7.ConclusionsPVR cells obtained from human membranes can be successfully isolated, cultured, and profiled in vitro. Using these primary cultures, we identified MTX as capable of significantly reducing growth and inducing cell death of PVR cells in vitro.

Evidence for pleural epithelial-mesenchymal transition in murine compensatory lung growth.

In many mammals, including rodents and humans, removal of one lung results in the compensatory growth of the remaining lung; however, the mechanism of compensatory lung growth is unknown. Here, we investigated the changes in morphology and phenotype of pleural cells after pneumonectomy. Between days 1 and 3 after pneumonectomy, cells expressing α-smooth muscle actin (SMA), a cytoplasmic marker of myofibroblasts, were significantly increased in the pleura compared to surgical controls (p < .01). Scanning electron microscopy of the pleural surface 3 days post-pneumonectomy demonstrated regions of the pleura with morphologic features consistent with epithelial-mesenchymal transition (EMT); namely, cells with disrupted intercellular junctions and an acquired mesenchymal (rounded and fusiform) morphotype. To detect the migration of the transitional pleural cells into the lung, a biotin tracer was used to label the pleural mesothelial cells at the time of surgery. By post-operative day 3, image cytometry of post-pneumonectomy subpleural alveoli demonstrated a 40-fold increase in biotin+ cells relative to pneumonectomy-plus-plombage controls (p < .01). Suggesting a similar origin in space and time, the distribution of cells expressing biotin, SMA, or vimentin demonstrated a strong spatial autocorrelation in the subpleural lung (p < .001). We conclude that post-pneumonectomy compensatory lung growth involves EMT with the migration of transitional mesothelial cells into subpleural alveoli.

Progressive Enrichment of Stemness Features and Tumor Stromal Alterations in Multistep Hepatocarcinogenesis.

Cancer stem cells (CSCs), a subset of tumor cells, contribute to an aggressive biological behavior, which is also affected by the tumor stroma. Despite the role of CSCs and the tumor stroma in hepatocellular carcinoma (HCC), features of stemness have not yet been studied in relation to tumor stromal alterations in multistep hepatocarcinogenesis. We investigated the expression status of stemness markers and tumor stromal changes in B viral carcinogenesis, which is the main etiology of HCC in Asia. Stemness features of tumoral hepatocytes (EpCAM, K19, Oct3/4, c-KIT, c-MET, and CD133), and tumor stromal cells expressing α-smooth muscle actin (α-SMA), CD68, CD163, and IL-6 were analyzed in 36 low grade dysplastic nodules (DNs), 48 high grade DNs, 30 early HCCs (eHCCs), and 51 progressed HCCs (pHCCs) by immunohistochemistry or real-time PCR. Stemness features (EpCAM and K19 in particular) were progressively acquired during hepatocarcinogenesis in combination with enrichment of stromal cells (CAFs, TAMs, IL-6+ cells). Stemness features were seen sporadically in DNs, more consistent in eHCCs, and peaked in pHCCs. Likewise, stromal cells were discernable in DNs, showed up as consistent cell densities in eHCCs and peaked in pHCCs. The stemness features and tumor stromal alterations also peaked in less differentiated or larger HCCs. In conclusion, progression of B viral multistep hepatocarcinogenesis is characterized by an enrichment of stemness features of neoplastic hepatocytes and a parallel alteration of the tumor stroma. The modulation of neoplastic hepatocytes and stromal cells was at low levels in precancerous lesions (DNs), consistently increased in incipient cancer (eHCCs) and peaked in pHCCs. Thus, in B viral hepatocarcinogenesis, interactions between CSCs and the tumor stroma, although starting early, seem to play a major role in tumor progression.

Targeting the Cardiac Myofibroblast Secretome to Treat Myocardial Fibrosis in Heart Failure.

Therapeutic strategies for heart failure (HF) should increasingly consider measures directly targeting the myocardium itself rather than strictly focusing on agents that unload the heart or target systemic neurohormones. In this regard, a new view is emerging that proposes the direct intervention on the pathological structural remodeling of the myocardium as part of HF therapy. One hallmark myocardial lesion in HF is the diffuse accumulation of collagen fibers (namely, collagen type I) within the interstitium and around the microvasculature of the myocardium (ie, myocardial fibrosis [MF]). MF is the result of the predominance of collagen synthesis over its degradation as a result of the actions of a population of persistent metabolically active cells: the myofibroblasts. MF impairs cardiac function and contributes to the development and worsening of HF, in addition to facilitating arrhythmias and ischemia, and thus adversely influences the evolution and outcome of nearly all cardiac diseases.1 Therefore, MF regression is an unmet medical need, and the search for new drugs aiming its reversal is active. However, the efficacy and safety of novel antifibrotic strategies currently under investigation seem questionable.2 We thus propose a therapeutic approach based on targeting the cardiac myofibroblast secretome with already available drugs, which have proven to be both efficient in reducing MF and safe in protecting cardiac function. In addition, we will discuss how to identify those HF patients in whom these therapies can be more beneficial and thus should be personalized. Cardiac myofibroblasts are cells expressing α-smooth muscle actin microfilaments that originate from the differentiation of several cell types (including resident cardiac fibroblasts) under the influence of many local and systemic factors (Figure 1). Myofibroblasts exhibit a synthetic phenotype that includes a secretome consisting of molecules requisite to modify the quantity and quality of the myocardial collagen network and facilitate MF …

Activation of endothelial NAD(P)H oxidase accelerates early glomerular injury in diabetic mice

Increased generation of reactive oxygen species (ROS) is a common denominative pathogenic mechanism underlying vascular and renal complications in diabetes mellitus. Endothelial NAD(P)H oxidase is a major source of vascular ROS, and it has an important role in endothelial dysfunction. We hypothesized that activation of endothelial NAD(P)H oxidase initiates and worsens the progression of diabetic nephropathy, particularly in the development of albuminuria. We used transgenic mice with endothelial-targeted overexpression of the catalytic subunit of NAD(P)H oxidase, Nox2 (NOX2TG). NOX2TG mice were crossed with Akita insulin-dependent diabetic (Akita) mice that develop progressive hyperglycemia. We compared the progression of diabetic nephropathy in Akita versus NOX2TG-Akita mice. NOX2TG-Akita mice and Akita mice developed significant albuminuria above the baseline at 6 and 10 weeks of age, respectively. Compared with Akita mice, NOX2TG-Akita mice exhibited higher levels of NAD(P)H oxidase activity in glomeruli, developed glomerular endothelial perturbations, and attenuated expression of glomerular glycocalyx. Moreover, in contrast to Akita mice, the NOX2TG-Akita mice had numerous endothelial microparticles (blebs), as detected by scanning electron microscopy, and increased glomerular permeability. Furthermore, NOX2TG-Akita mice exhibited distinct phenotypic changes in glomerular mesangial cells expressing α-smooth muscle actin, and in podocytes expressing increased levels of desmin, whereas the glomeruli generated increased levels of ROS. In conclusion, activation of endothelial NAD(P)H oxidase in the presence of hyperglycemia initiated and exacerbated diabetic nephropathy characterized by the development of albuminuria. Moreover, ROS generated in the endothelium compounded glomerular dysfunctions by altering the phenotypes of mesangial cells and compromising the integrity of the podocytes.

Mechanisms underlying spontaneous constrictions of postcapillary venules in the rat stomach.

Postcapillary venules (PCVs) play a critical role in regulating capillary hydrostatic pressure, but their contractile mechanisms are not well understood. We examined the properties of spontaneous vasomotion and corresponding Ca(2+) transients in gastric PCV. In the rat gastric submucosa, changes in PCV diameter and intracellular Ca(2+) dynamics were visualised by video tracking system and fluorescent Ca(2+) imaging, respectively, while PCV morphology was examined by immunohistochemistry. Stellate-shaped PCV mural cells expressing α-smooth muscle actin exhibited synchronised spontaneous Ca(2+) transients to develop vasomotion which was abolished by nifedipine (1 μM), cyclopiazonic acid (10 μM), or Ca(2+)-activated Cl(-) channel inhibitors (100 μM niflumic acid, 1 μM T16Ainh-A01). A gap junction blocker (3 μM carbenoxolone) disrupted the synchrony of spontaneous Ca(2+) transients amongst PCV mural cells and attenuated spontaneous vasomotion. Low chloride solution ([Cl(-)]0 = 12.4 mM) also disrupted the synchrony of spontaneous Ca(2+) transients and abolished vasomotion. Na(+)-K(+)-Cl(-) co-transporter inhibitors (10 μM bumetanide, 30 μM furosemide) suppressed spontaneous Ca(2+) transients and vasoconstrictions. A phosphodiesterase type 5 (PDE5) inhibitor (1 μM tadalafil) disrupted the spontaneous Ca(2+) transient synchrony and abolished vasomotion in a nitric oxide (NO)-dependent manner. Thus, gastric PCVs exhibit spontaneous vasomotion, resulting from synchronised spontaneous Ca(2+) transients within a network of stellate-shaped PCV mural cells. An active Cl(-) accumulation partly via Na(+)-K(+)-Cl(-) co-transport appears to be fundamental in maintaining depolarisation upon the opening of Ca(2+)-activated Cl(-) channels that triggers Ca(2+) influx via voltage-dependent L-type Ca(2+) channels. Basal PDE5 activity may continuously counteract vaso-relaxing effects of endothelial NO to maintain spontaneous vasomotion.

Effect of local injection of mesenchymal stem cells on healing of sutured gastric perforation in an experimental model.

Mesenchymal stem cells are proposed to facilitate repair of organ injuries. The aim of this study was to investigate whether local injection of mesenchymal stem cells could accelerate healing of sutured gastric perforations. Sutured gastric perforations in rats were treated either with local injection of mesenchymal stem cells (injected MSC group) or by topically spraying with fibrin glue containing mesenchymal stem cells (sprayed MSC group). Controls were treated by local injection of saline or topical spray of fibrin glue without mesenchymal stem cells. Healing of sutured gastric perforations was assessed on days 3, 5 and 7. Local injection of mesenchymal stem cells significantly promoted the healing of gastric perforations, with the highest pneumatic bursting pressure (mean(s.e.m.) 112·3(30·2) mmHg on day 5 versus 71·2(17·4) mmHg in saline controls; P = 0·001), minimal wound adhesions, and lowest incidence of wound dehiscence (3, 6, 5 and 1 animal on day 5 in control, fibrin, sprayed MSC and injected MSC groups respectively; n = 10 per group) and abdominal abscess (2, 2, 1 and no animals respectively on day 5). Histological examination showed that gastric perforations in the injected MSC group displayed reduced inflammation, and increased granulation and re-epithelialization. Sutured gastric perforations in the injected MSC group showed decreased expression of interleukin 6, and increased expression of transforming growth factor β1 and epithelial proliferating cell nuclear antigen, compared with the other groups. Local injection of mesenchymal stem cells was more effective than topical application, and enhanced the healing of sutured gastric perforations by an anti-inflammatory process, enhanced cellular proliferation and earlier onset of granulation. Surgical relevance Abnormal healing of gastric perforation may cause morbidity and increase the risk of death. Adipose tissue-derived mesenchymal stem cells have been found to promote the healing of organ injuries through cellular differentiation and secretion of cytokines that stimulate cellular proliferation and angiogenesis, and suppress inflammation. This study explored the therapeutic potential of such mesenchymal stem cells for promotion of the healing of sutured gastric perforations. Mesenchymal stem cells delivered by local injection significantly enhanced the healing of gastric perforations with reduced severity of wound adhesion, and a decreased incidence of wound dehiscence and abdominal abscess. The increased expression of transforming growth factor β1, proliferating cell nuclear antigen and reduced level of interleukin 6 provide evidence for enhancement of the healing process. Engrafted mesenchymal stem cells expressed α-smooth muscle actin as a marker of myofibroblasts. This preclinical study indicates that local injection of allogeneic adipose tissue-derived mesenchymal stem cells may have a potential therapeutic role in enhancing the healing of peptic ulcer disease and prevention of ulcer-related complications.

The kampo medicine Daikenchuto inhibits peritoneal fibrosis in mice.

Long-term peritoneal dialysis therapy causes inflammation and histological changes in the peritoneal membrane. Inflammation generally activates fibroblasts and results in fibroblast-myofibroblast differentiation. Heat-shock protein 47 (HSP 47), a collagen-specific molecular chaperone, is localized in myofibroblasts and is involved in the progression of peritoneal fibrosis. Daikenchuto (DKT), a Kampo medicine, is used to prevent postoperative colon adhesion. It inhibits inflammation and HSP 47 expression in the gastrointestinal tract. We examined the effect of DKT on chlorhexidine gluconate (CG)-induced peritoneal fibrosis in mice injected with 0.1% CG dissolved in 15% ethanol. DKT was dissolved in the drinking water. Histological changes were assessed using Masson trichrome staining. Cells expressing α-smooth muscle actin (α-SMA), HSP 47, phospho-Smad 2/3, F4/80, and monocyte chemotactic protein-1 were examined immunohistochemically. Compared with the control group, the peritoneal tissues of the CG group were markedly thickened, and the number of cells expressing α-SMA, HSP 47, phospho-Smad 2/3, F4/80, and monocyte chemotactic protein-1 was significantly increased. However, these changes were inhibited in the DKT-treated group. These results indicate that DKT can prevent peritoneal fibrosis by inhibiting inflammation and HSP 47 expression.

Recruitment and subsequent proliferation of bone marrow-derived cells in the postischemic kidney are important to the progression of fibrosis.

Acute kidney injury (AKI) is an independent risk factor of the development of chronic kidney disease. Kidney fibrosis is a typical feature of chronic kidney disease and is characterized as an expansion of the interstitium due to increases in extracellular matrix molecules and interstitial cells caused by accumulations of extrarenal cells and by the proliferation or differentiation of intrarenal cells. However, the role of bone marrow-derived cells (BMDCs) in AKI-induced kidney fibrosis remains to be defined. Here, we investigated the role of BMDCs in kidney fibrosis after ischemia-reperfusion injury (IRI)-induced AKI in green fluorescent protein (GFP)-expressing bone marrow chimeric mice. IRI resulted in severe fibrotic changes in kidney tissues and dramatically increased interstitial cell numbers. Furthermore, GFP-expressing BMDCs accounted for >80% of interstitial cells in fibrotic kidneys. Interstitial GFP-expressing cells expressed α-smooth muscle actin (a myofibroblast marker), fibroblast-specific protein-1 (a fibroblast marker), collagen type III, and F4/80 (a macrophage marker). Over 20% of interstitial cells were bromodeoxyuridine-incorporating (proliferating) cells, and of these, 80% cells were GFP-expressing BMDCs. Daily treatment of IRI mice with apocynin (a NADPH oxidase inhibitor that functions as an antioxidant) from the day after surgery until euthanization slightly inhibited these changes with a small reduction of fibrosis. Taken together, our findings show that BMDCs make a major contribution to IRI-induced fibrosis due to their infiltration, subsequent differentiation, and proliferation in injured kidneys, suggesting that BMDCs be considered an important target for the treatment of kidney fibrosis.

The expression of groups IIE and V phospholipase A2 is associated with an increased expression of osteogenic molecules in human calcified aortic valves.

Eicosanoids play various pathogenic roles in aortic valve calcification. Eicosanoids are derived from the arachidonic acid generated by phospholipase A2 (PLA2). We therefore sought to determine whether PLA2s are expressed in human aortic valves and, if so, whether the expression of PLA2s is related to the expression of osteogenic molecules in these tissues. Histological and gene expression analyses of 38 non-rheumatic aortic valves obtained at the time of cardiac valve replacement surgery were conducted. Moreover, gene expression analyses were performed using valve interstitial cells (VICs) obtained from human aortic valves. Among the PLA2s examined, the degree of immunoreactivity for PLA2s-IIE and -V was found to significantly correlate with the grade of calcification in the aortic valves. The degree of immunoreactivity and gene expression levels of PLA2s-IIE and -V significantly correlated with those of bone morphogenetic protein (BMP)-2, osteopontin and alkaline phosphatase (ALP). In addition, immunoreactivity for cyclooxygenase (COX)-1, COX-2 and 5-lipoxygenase, downstream enzymes of PLA2 in the arachidonic acid cascade, was co-localized with that for PLA2s-IIE and -V in cells expressing α-smooth muscle actin and macrophages expressing CD68. Furthermore, in the in vitro experiments using cultured VICs, the mRNA expression levels of BMP-2, osteopontin and ALP were suppressed by the inhibition of the expression of PLA2s-IIE or -V with specific siRNAs. The expression of PLA2s-IIE and -V correlates with the development of calcification as well as the expression of pro-osteogenic molecules in human aortic valves, and inhibiting the expression of PLA2s-IIE and -V suppresses the induction of osteogenic molecules in cultured cells. Therefore, PLA2s-IIE and -V may play a role in the pathogenesis of valve calcification.

Liver precursor cells increase hepatic fibrosis induced by chronic carbon tetrachloride intoxication in rats

Hepatic fibrosis, the major complication of virtually all types of chronic liver damage, usually begins in portal areas, and its severity has been correlated to liver progenitor cells (LPC) expansion from periportal areas, even if the primary targets of injury are intralobular hepatocytes. The aim of this study was to determine the potential fibrogenic role of LPC, using a new experimental model in which rat liver fibrosis was induced by chronic carbon tetrachloride (CCl4) administration for 6 weeks, in combination with chronic acetylaminofluorene treatment (AAF), which promotes activation of LPC compartment. Treatment with CCl4 alone caused a significant increase in serum transaminase activity as well as liver fibrosis initiating around central veins and leading to formation of incomplete centro-central septa with sparse fibrogenic cells expressing α-smooth muscle actin (αSMA). In AAF/CCl4-treated animals, the fibrogenic response was profoundly worsened, with formation of multiple porto-central bridging septa leading to cirrhosis, whereas hepatocellular necrosis and inflammation were similar to those observed in CCl4-treated animals. Enhanced fibrosis in AAF/CCl4 group was accompanied by ductule forming LPC expanding from portal areas, αSMA-positive cells accumulation in the fibrotic areas and increased expression of hepatic collagen type 1, 3 and 4 mRNA. Moreover, CK19-positive LPC expressed the most potent fibrogenic cytokine transforming growth factor-β (TGFβ) without any expression of αSMA, desmin or fibroblast-specific protein-1, demonstrating that LPC did not undergo an epithelial–mesenchymal transition. In this new experimental model, LPC, by expressing TGFβ, contributed to the accumulation of αSMA-positive myofibroblasts in the ductular reaction leading to enhanced fibrosis but also to disease progression and to a fibrotic pattern similar to that observed in humans.

Host-Derived Pericytes and Sca-1+Cells Predominate in the MART-1−Stroma Fraction of Experimentally Induced Melanoma

Identification of cell types in tumor-associated stroma that are involved in the development of melanoma is hampered by their heterogeneity. The authors used flow cytometry and immunohistochemistry to demonstrate that anti-MART-1 antibodies can discriminate between melanoma and stroma cells. They investigated the cellular composition of the MART-1-, non-hematopoietic melanoma-associated stroma, finding it consisted mainly of Sca-1+ and CD146+ cells. These cell types were also observed in the skin and muscle adjacent to developing melanomas. The Sca-1+ cell population was observed distributed in the epidermis, hair follicle bulges, and tumor capsule. The CD146+ population was found distributed within the tumor, mainly associated with blood vessels in a perivascular location. In addition to a perivascular distribution, CD146+ cells expressed α-smooth muscle actin, lacked expression of endothelial markers CD31 and CD34, and were therefore identified as pericytes. Pericytes were found to be associated with CD31+ endothelial cells; however, some pericytes were also observed associated with CD31-, MART-1+ B16 melanoma cells that appeared to form blood vessel structures. Furthermore, the authors observed extensive nuclear expression of HIF-1α in melanoma and stroma cells, suggesting hypoxia is an important factor associated with the melanoma microenvironment and vascularization. The results suggest that pericytes and Sca-1+ stroma cells are important contributors to melanoma development.

Reciprocal Induction of Simple Organogenesis by Mouse Kidney Progenitor Cells in Three-Dimensional Co-Culture

Kidney development is regulated by a coordinated reciprocal induction of metanephric mesenchymal (MM) and ureteric bud (UB) cells. Here, established MM and UB progenitor cell lines were recombined in three-dimensional Matrigel implants in SCID mice. Differentiation potential was examined for changes in phenotype, organization, and the presence of specialized proteins using immunofluorescence and bright-field and electron microscopy. Both cell types, when grown alone, did not develop into specialized structures. When combined, the cells organized into simple organoid structures of polarized epithelia with lumens surrounded by capillary-like structures. Tracker experiments indicated the UB cells formed the tubuloid structures, and the MM cells were the source of the capillary-like cells. The epithelial cells stained positive for pancytokeratin, the junctional complex protein ZO-1, collagen type IV, as well as UB and collecting duct markers, rearranged during transfection (RET), Dolichos biflorus lectin, EndoA cytokeratin, and aquaporin 2. The surrounding cells expressed α-smooth muscle actin, vimentin, platelet endothelial cell adhesion molecule 1 (PECAM), and aquaporin 1, a marker of vasculogenesis. The epithelium exhibited apical vacuoles, microvilli, junctional complexes, and linear basement membranes. Capillary-like structures showed endothelial features with occasional pericytes. UB cell epithelialization was augmented in the presence of MM cell-derived conditioned medium, glial-derived neurotrophic factor (GDNF), hepatocyte growth factor (HGF), or fibronectin. MM cells grown in the presence of UB-derived conditioned medium failed to undergo differentiation. However, UB cell-derived conditioned medium induced MM cell migration. These studies indicate that tubulogenesis and vasculogenesis can be partially recapitulated by recombining individual MM and UB cell lineages, providing a new model system to study organogenesis ex vivo.

Simvastatin inhibits sphingosylphosphorylcholine-induced differentiation of human mesenchymal stem cells into smooth muscle cells

Sphingosylphosphorylcholine (SPC) induces differentiation of human adipose tissue-derived mesenchymal stem cells (hASCs) into smooth muscle-like cells expressing α-smooth muscle actin (α-SMA) via transforming growth factor-β1/Smad2- and RhoA/Rho kinase-dependent mechanisms. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins) have been known to have beneficial effects in the treatment of cardiovascular diseases. In the present study, we examined the effects of simvastatin on the SPC-induced α-SMA expression and Smad2 phosphorylation in hASCs. Simvastatin inhibited the SPC-induced α-SMA expression and sustained phosphorylation of Smad2 in hASCs. SPC treatment caused RhoA activation via a simvastatin-sensitive mechanism. The SPC-induced α-SMA expression and Smad2 phosphorylation were abrogated by pretreatment of the cells with the Rho kinase inhibitor Y27632 or overexpression of a dominant negative RhoA mutant. Furthermore, SPC induced secretion of TGF-β1 and pretreatment with either Y27632 or simvastatin inhibited the SPC-induced TGF-β1 secretion. These results suggest that simvastatin inhibits SPC-induced differentiation of hASCs into smooth muscle cells by attenuating the RhoA/Rho kinase-dependent activation of autocrine TGF-β1/Smad2 signaling pathway.