Cell sex affects extracellular matrix protein expression and proliferation of smooth muscle progenitor cells derived from human pluripotent stem cells.

BACKGROUND: Smooth muscle progenitor cells (pSMCs) differentiated from human pluripotent stem cells (hPSCs) hold great promise for treating diseases or degenerative conditions involving smooth muscle pathologies. However, the therapeutic potential of pSMCs derived from men and women may be very different. Cell sex can exert a profound impact on the differentiation process of stem cells into somatic cells. In spite of advances in translation of stem cell technologies, the role of cell sex and the effect of sex hormones on the differentiation towards mesenchymal lineage pSMCs remain largely unexplored. METHODS: Using a standard differentiation protocol, two human embryonic stem cell lines (one male line and one female line) and three induced pluripotent stem cell lines (one male line and two female lines) were differentiated into pSMCs. We examined differences in the differentiation of male and female hPSCs into pSMCs, and investigated the effect of 17ß-estradiol (E2) on the extracellular matrix (ECM) metabolisms and cell proliferation rates of the pSMCs. Statistical analyses were performed by using Student's t test or two-way ANOVA, p < 0.05. RESULTS: Male and female hPSCs had similar differentiation efficiencies and generated morphologically comparable pSMCs under a standard differentiation protocol, but the derived pSMCs showed sex differences in expression of ECM proteins, such as MMP-2 and TIMP-1, and cell proliferation rates. E2 treatment induced the expression of myogenic gene markers and suppressed ECM degradation activities through reduction of MMP activity and increased expression of TIMP-1 in female pSMCs, but not in male pSMCs. CONCLUSIONS: hPSC-derived pSMCs from different sexes show differential expression of ECM proteins and proliferation rates. Estrogen appears to promote maturation and ECM protein expression in female pSMCs, but not in male pSMCs. These data suggest that intrinsic cell-sex differences may influence progenitor cell biology.

Characterization of interstitial Cajal progenitors cells and their changes in Hirschsprung's disease.

Interstitial cells of Cajal (ICC) are critical to gastrointestinal motility. The phenotypes of ICC progenitors have been observed in the mouse gut, but whether they exist in the human colon and what abnormal changes in their quantity and ultrastructure are present in Hirschsprung's disease (HSCR) colon remains uncertain. In this study, we collected the surgical resection of colons, both proximal and narrow segments, from HSCR patients and normal controls. First, we identified the progenitor of ICC in normal adult colon using immunofluorescent localization techniques with laser confocal microscopy. Next, the progenitors were sorted to observe their morphology. We further applied flow cytometry to examine the content of ICC progenitors in these fresh samples. The ultrastructural changes in the narrow and proximal parts of the HSCR colon were observed using transmission electron microscopy (TEM) and were compared with the normal adult colon. The presumed early progenitor (c-Kit(low)CD34(+)Igf1r(+)) and committed progenitor (c-Kit(+)CD34(+)Igf1r(+)) of ICC exist in adult normal colon as well as in the narrow and proximal parts of the HSCR colon. However, the proportions of mature, early and committed progenitors of ICC were dramatically reduced in the narrow segment of the HSCR colon. The proportions of mature and committed progenitors of ICC in the proximal segment of the HSCR colon were lower than in the adult normal colon. Ultrastructurally, ICC, enteric nerves, and smooth muscle in the narrow segment of the HSCR colon showed severe injury, including swollen vacuola or ted mitochondria, disappearance of mitochondrial cristae, dilated rough endoplasmic reticulum, vesiculation and degranulation, and disappearance of the caveolae on the ICC membrane surface. The contents of ICC and its progenitors in the narrow part of the HSCR colon were significantly decreased than those of adult colon, which may be associated with HSCR pathogenesis.

Genetically modified human myoblasts with eNOS may improve regenerative ability of myogenic stem cells to infarcted heart.

BACKGROUND: Modern therapies of post infarcted heart failure are focused on perfusion improvement of the injured myocardium. This effect can be achieved by, among other means, implanting stem cells which could be genetically modified with factors inducing the formation of new blood vessels in the post infarction scar area. Combined stem cell and gene therapy seems to be a promising strategy to heal an impaired myocardium. The creation of new blood vessels can be indirectly stimulated via factors inducing vascular endothelial growth factor synthesis, for example endothelial nitric oxide synthase (eNOS). The product of this enzyme, nitric oxide, is a molecule that can influence numerous physiological activities; it can contribute to vasodilation, stimulation of endothelial cell growth, prevention of platelet aggregation and leukocyte adhesion to the endothelium. AIM: To verify the pro-angiogenic and regenerative potential of human primary myoblasts and murine myoblast cell line C2C12 transiently transfected with eNOS gene. METHODS: Stem cells (either human or murine) were maintained in standard in vitro conditions. Next, both types of myoblasts were modified using electroporation and lipofection (human and murine cells), respectively. The efficacy of the transfection method was evaluated using flow cytometry. The concentration of eNOS protein was measured by ELISA immunoassay. The biological properties of modified cells were assessed using an MTT proliferation test and DAPI cell cycle analysis. To verify the influence of oxidative stress on myoblasts, cytometric tests using Annexin V and propidium iodide were applied. To check possible alterations in myogenic gene expression of stem cells transduced by genetic modification, the myogenic regulatory factors were evaluated by real-time PCR. The function of genetic modification was confirmed by a HUVEC capillary sprouting test using myoblasts supernatants. RESULTS: Electroporation turned out to be an efficient transfection method. High amounts of secreted protein were obtained (in the range 2,000 pg/mL) in both cell types studied. Moreover, the functionality of gene overexpression product was confirmed in capillary development assay. Human myoblasts did not exhibit any changes in cell cycle; however, eNOS transfected murine myoblasts revealed a statistically significant reduction in cell cycle ratio compared to controls (p < 0.001). In the case of myogenic gene expression, a decrease in Myogenin level was only detected in the human transfected myoblast population (p < 0.05). CONCLUSIONS: The results of our study may suggest that transplantation of myoblasts overexpressing eNOS could be promising for cell therapy in regenerating the post infarction heart.

Tissue engineered esophagus by mesenchymal stem cell seeding for esophageal repair in a canine model.

PURPOSE: Acellular porcine small intestinal submucosa (SIS) has been successfully used for esophagoplasty in dogs. However, this has not led to complete epithelialization and muscular regeneration. We undertook the present study to assess the effect of tissue-engineered esophagus generated by seeding bone marrow mesenchymal stem cells (BMSCs) onto an SIS scaffold (BMSCs-SIS) in a canine model. METHODS: We cultured, passaged, and measured autologous BMSCs and myoblasts with cell proliferation and immunohistochemical assays. We labeled the third passage of BMSCs with PKH-26, a fluorescent dye, before seeded it onto the SIS. We resected canine cervical esophagus to generate a defect 5 cm in length and 50% in circumference, which we repaired with BMSCs-SIS or SIS alone. RESULTS: Four weeks later, barium esophagram demonstrated that esophageal lumen surface of the patch graft was smoother in the BMSCs-SIS group compared with the SIS group. Histological examination suggested a strong similarity between BMSCs and esophageal myoblasts in terms of morphology and function. Although both BMSCs-SIS and SIS repaired the esophageal defects, we noted complete re-epithelialization with almost no inflammation only in the former group. By 12 wk after the surgery, we observed long bundles of skeletal muscles only in the BMSCs-SIS group, where the microvessel density was also much greater. CONCLUSIONS: Bone marrow mesenchymal stem cells on an SIS scaffold can promote re-epithelialization, revascularization, and muscular regeneration. This approach may provide an attractive option for esophageal regeneration.

The arterial and cardiac epicardium in development, disease and repair.

The importance of the epicardium covering the heart and the intrapericardial part of the great arteries has reached a new summit. It has evolved as a major cellular component with impact both in development, disease and more recently also repair potential. The role of the epicardium in development, its differentiation from a proepicardial organ at the venous pole (vPEO) and the differentiation capacities of the vPEO initiating cardiac epicardium (cEP) into epicardium derived cells (EPDCs) have been extensively described in recent reviews on growth and transcription factor pathways. In short, the epicardium is the source of the interstitial, the annulus fibrosus and the adventitial fibroblasts, and differentiates into the coronary arterial smooth muscle cells. Furthermore, EPDCs induce growth of the compact myocardium and differentiation of the Purkinje fibers. This review includes an arterial pole located PEO (aPEO) that provides the epicardium covering the intrapericardial great vessels. In avian and mouse models disturbance of epicardial outgrowth and maturation leads to a broad spectrum of cardiac anomalies with main focus on non-compaction of the myocardium, deficient annulus fibrosis, valve malformations and coronary artery abnormalities. The discovery that in disease both arterial and cardiac epicardium can again differentiate into EPDCs and thus reactivate its embryonic program and potential has highly broadened the scope of research interest. This reactivation is seen after myocardial infarction and also in aneurysm formation of the ascending aorta. Use of EPDCs for cell therapy show their positive function in paracrine mediated repair processes which can be additive when combined with the cardiac progenitor stem cells that probably share the same embryonic origin with EPDCs. Research into the many cell-autonomous and cell-cell-based capacities of the adult epicardium will open up new realistic therapeutic avenues.

The smooth muscle microRNA miR-145 regulates gut epithelial development via a paracrine mechanism.

MicroRNAs are potent modulators of cellular differentiation. miR-145 is expressed in, and promotes the differentiation of vascular and visceral smooth muscle cells (SMCs). Interestingly, we have observed that miR-145 also promotes differentiation of the gut epithelium in the developing zebrafish, a cell type where it is not expressed. Here we identify that a paracrine pathway involving the morphogens Sonic hedgehog (Shh) in epithelium and bone morphogenic protein 4 (Bmp4) in SMCs is modulated by miR-145. We show that expression of miR-145 in visceral SMCs normally represses the expression of the morphogen bmp4, as loss of miR-145 leads to upregulation of bmp4 in SMCs. We show that bmp4 in turn controls expression of Shh in the visceral epithelium. Conversely, in miR-145 morphants where bmp4 expression is increased, expression of sonic hedgehog a (shha) is strongly increased in gut epithelium. We show that expression of bmp4 is modulated by the miR-145 direct target gata6 but not a second potential direct target, klf5a. Thus although miR-145 is a tissue-restricted microRNA, it plays an essential role in promoting the patterning of both gut layers during gut development via a paracrine mechanism.

Differentiation of vascular smooth muscle cells from local precursors during embryonic and adult arteriogenesis requires Notch signaling.

Vascular smooth muscle cells (VSMC) have been suggested to arise from various developmental sources during embryogenesis, depending on the vascular bed. However, evidence also points to a common subpopulation of vascular progenitor cells predisposed to VSMC fate in the embryo. In the present study, we use binary transgenic reporter mice to identify a Tie1(+)CD31(dim)vascular endothelial (VE)-cadherin(-)CD45(-) precursor that gives rise to VSMC in vivo in all vascular beds examined. This precursor does not represent a mature endothelial cell, because a VE-cadherin promoter-driven reporter shows no expression in VSMC during murine development. Blockade of Notch signaling in the Tie1(+) precursor cell, but not the VE-cadherin(+) endothelial cell, decreases VSMC investment of developing arteries, leading to localized hemorrhage in the embryo at the time of vascular maturation. However, Notch signaling is not required in the Tie1(+) precursor after establishment of a stable artery. Thus, Notch activity is required in the differentiation of a Tie1(+) local precursor to VSMC in a spatiotemporal fashion across all vascular beds.

Transforming growth factor-ß signaling in myogenic cells regulates vascular morphogenesis, differentiation, and matrix synthesis.

OBJECTIVE: Transforming growth factor-ß (TGF-ß) signaling is required for normal vascular development. We aimed to discover the role of TGF-ß signaling in embryonic smooth muscle cells (SMCs). METHODS AND RESULTS: We bred mice with smooth muscle (SM) 22α-Cre and Tgfbr2(flox) alleles to generate embryos in which the type II TGF-ß receptor (TGFBR2; required for TGF-ß signaling) was deleted in SMCs. Embryos were harvested between embryonic day (E) 9.5 and E18.5 and examined grossly, microscopically, and by histochemical and RNA analyses. SM22α-Cre(+/0) Tgfbr2(flox/flox) (knockout [KO]) embryos died before E15.5 with defects that included cardiac outflow tract abnormalities, persistence of the right dorsal aorta, and dilation of the distal aorta. Histological analyses suggested normal expression of SMC differentiation markers in KO aortas; however, RNA analyses showed that SMC differentiation markers were increased in KO cardiac outflow vessels but decreased in the descending aorta. KO aortas had only rare mature elastin deposits and contained abnormal aggregates of extracellular matrix proteins. Expression of several matrix proteins was significantly decreased in KO descending aortas but not in cardiac outflow vessels. CONCLUSIONS: TGF-ß signaling in SMCs controls differentiation, matrix synthesis, and vascular morphogenesis. Effects of TGF-ß on SMC gene expression appear to differ depending on the location of SMCs in the aorta.

Distant cis-regulatory elements in human skeletal muscle differentiation.

Identifying gene regulatory elements and their target genes in human cells remains a significant challenge. Despite increasing evidence of physical interactions between distant regulatory elements and gene promoters in mammalian cells, many studies consider only promoter-proximal regulatory regions. We identify putative cis-regulatory modules (CRMs) in human skeletal muscle differentiation by combining myogenic TF binding data before and after differentiation with histone modification data in myoblasts. CRMs that are distant (>20 kb) from muscle gene promoters are common and are more likely than proximal promoter regions to show differentiation-specific changes in myogenic TF binding. We find that two of these distant CRMs, known to activate transcription in differentiating myoblasts, interact physically with gene promoters (PDLIM3 and ACTA1) during differentiation. Our results highlight the importance of considering distal CRMs in investigations of mammalian gene regulation and support the hypothesis that distant CRM-promoter looping contacts are a general mechanism of gene regulation.

Resident vascular progenitor cells.

Homeostasis of the vessel wall is essential for maintaining its function, including blood pressure and patency of the lumen. In physiological conditions, the turnover rate of vascular cells, i.e. endothelial and smooth muscle cells, is low, but markedly increased in diseased situations, e.g. vascular injury after angioplasty. It is believed that mature vascular cells have an ability to proliferate to replace lost cells normally. On the other hand, recent evidence indicates stem/progenitor cells may participate in vascular repair and the formation of neointimal lesions in severely damaged vessels. It was found that all three layers of the vessels, the intima, media and adventitia, contain resident progenitor cells, including endothelial progenitor cells, mesenchymal stromal cells, Sca-1+ and CD34+ cells. Data also demonstrated that these resident progenitor cells could differentiate into a variety of cell types in response to different culture conditions. However, collective data were obtained mostly from in vitro culture assays and phenotypic marker studies. There are many unanswered questions concerning the mechanism of cell differentiation and the functional role of these cells in vascular repair and the pathogenesis of vascular disease. In the present review, we aim to summarize the data showing the presence of the resident progenitor cells, to highlight possible signal pathways orchestrating cell differentiation toward endothelial and smooth muscle cells, and to discuss the data limitations, challenges and controversial issues related to the role of progenitors. This article is part of a special issue entitled, "Cardiovascular Stem Cells Revisited".

Circulating smooth muscle progenitor cells in arterial remodeling.

The proliferation and migration of vascular smooth muscle cells (SMCs) from the media toward the intimal layer are key components in vascular proliferative diseases. In addition, the differentiation of circulating bone marrow-derived mononuclear cells (BMMCs) into SMCs has been described to contribute to lesion progression in experimental models of atherosclerosis, transplant arteriosclerosis, and neointima formation. In vitro, CD14(+) BMMCs from peripheral blood acquire a spindle-shaped phenotype and express specific SMC markers in response to platelet-derived growth factor-BB. However, the 'trans-differentiation' capacity of BMMCs into definitive SMCs in vivo remains a highly controversial issue. Whereas SMCs within atherosclerotic plaques have been demonstrated to be exclusively of local origin, more severe injury models have shown a wide diversity of SMCs or smooth muscle-like cells derived from BMMCs. In hindsight, these discrepancies may be attributed to methodological differences, e.g., the use of high-resolution microscopy or the specificity of the SMC marker proteins. In fact, the analysis of mouse strains that express marker genes under the control of a highly specific smooth muscle-myosin heavy chain (SM-MHC) promoter and a time-course analysis on the dynamic process of neointima formation have recently shown that BMMCs temporarily express α-smooth muscle actin, not SM-MHC. Additionally, BM-derived cells disappear from the neointimal lesion after the inflammatory response to the injury has subsided. Although CD14(+)/CD68(+) have important paracrine effects on arterial lesion progression, BMMCs account for more of the 'SMC-like macrophages' than the highly 'trans-differentiated' and definitive SMCs in vivo. This article is part of a special issue entitled, "Cardiovascular Stem Cells Revisited".

[Comparison of myogenic differentiation ability of adipose-derived stem cells from different sites in rabbit].

OBJECTIVE: To compare the myogenic differentiation ability in vitro of rabbit adipose-derived stem cells (ADCSs) from different sites so as to provide ideal seed cells for repair and reconstruction of urinary tract. METHODS: Adipose tissues were obtained from the nape of the neck, post peritoneum, and vicinity of epididymis of a 4-month-old male New Zealand rabbit and ADSCs were harvested through collagenase digestion. ADSCs were purified by differential attachment method. The protein marker CD44 of rabbit ADSCs was used to identify the stem cells by immunocytochemistry, then the 5th generation of ADSCs were induced to differentiate into adipogenic, osteogenic, and myogenic cells. Multi-differentiation was confirmed by Oil red O staining, von Kossa staining, and RT-PCR. Myogenic differentiation abilities of ADSCs from 3 different sites were compared between the control group (L-DMEM medium containing 10%FBS) and the experimental group (myogenic medium) by RT-PCR method. RESULTS: ADSCs could be easily isolated from adipose tissues of the nape of the neck, post peritoneum, and vicinity of epididymis. ADSCs displayed a typical cobblestone morphology. Brown particles could be seen in ADSCs by CD44 immunocytochemistry staining. Oil red O staining showed red fat drops in ADSCs after 14 days of adipogenic culture. Black matrix could be seen in ADSCs by von Kossa staining after 28 days of osteogenic culture. RT-PCR detection showed moderate alpha-actin expression in the control group and strong alpha-actin expression in the experimental group after 42 days of myogenic culture. The growth rate of alpha-actin from the adipose tissue of post peritoneum (28.622% +/- 4.879%) was significantly lower (P < 0.05) than those from the adipose tissues of the nape of the neck (35.471% +/- 3.434%) and vicinity of epididymis (38.446% +/- 4.852%). CONCLUSION: The ADSCs from different sites show different myogenic differentiation abilities in vitro. ADSCs from the adipose tissues of the nape of the neck and vicinity of epididymis can be used as ideal seed cells for tissue engineering of lower urinary tract.

Induced differentiation of adipose-derived stromal cells into myoblasts.

This study aimed to induce the differentiation of isolated and purified adipose-derived stromal cells (ADSCs) into myoblasts, which may provide a new strategy for tissue engineering in patients with stress urinary incontinence (SUI). ADSCs, isolated and cultured ex vivo, were identified by flow cytometry and induced to differentiate into myoblasts in the presence of an induction solution consisting of DMEM supplemented with 5-azacytidine (5-aza), 5% FBS, and 5% horse serum. Cellular morphology was observed under an inverted microscope. Ultrastructural changes occurring during the differentiation were observed by transmission electron microscopy and confocal laser scanning microscopy. Cellular immunohistochemical staining was applied to determine the expression of desmin protein in cells with and without induced differentiation. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to detect mRNA and protein expression, respectively, of sarcomeric and desmin smooth muscle proteins. The results showed that ADSCs were mainly of a spindle or polygon shape. Flow cytometry analysis revealed that ADSCs did not express CD34, CD45, and CD106 but high levels of CD44 and CD90, which confirmed that the cultured cells were indeed ADSCs. After induction with a 5-aza-containing solution, morphological changes in ADSCs, including irregular cell size, were observed. Cells gradually changed from long spindles to polygons and star-shaped cells with microvilli on the cell surface. Many organelles were observed and the cytoplasm was found to contain many mitochondria, rough endoplasmic reticulum (rER), and myofilament-like structures. Cell immunohistochemical staining revealed different levels of desmin expression in each phase of the induction process, with the highest expression level found on day 28 of induction. RT-PCR and Western blot results confirmed significantly higher desmin gene expression in induced cells compared with control cells, but no significant difference between the two groups of cells in sarcomeric protein expression. It was concluded that under specific induction setting, ADSCs can be induced to differentiate into myoblasts, providing a potential new option in stem cell transplantation therapy for SUI.

Isolation and characterization of bovine intestinal subepithelial myofibroblasts.

Intestinal subepithelial myofibroblasts (ISMFs) are mesenchymal cells that exist under the epithelium of intestines. Primarily isolated ISMFs from rodents have been applied to experiments. However, due to the size of their intestines, the available cell number is limited. Thus, we attempted to isolate ISMFs from bovine colon as an alternative material. After detachment of smooth muscle and epithelial layers, colonic mucosa was explanted. After 2-week incubation, alpha-SMA+ / vimentin+ / desmin(-) ISMFs were harvested and applied for experiments. First we examined the effect of cell passage on morphology and proliferation activity of bovine ISMFs. Although 3rd and 7th passage bovine ISMFs did not exhibit any changes, 11th passage ISMFs showed rounded enlarged shape and lost proliferation potential. On the contrary, rat ISMFs displayed the above senescent changes at earlier passage (passage 4). In intracellular Ca2+ concentration measurement, bioactive substances (0.3-1 microM ATP, 0.1-1 microM serotonin, 10-100 nM endothelin-1, and 1-10 nM bradykinin) dose-dependently induced an increase in intracellular Ca2+ concentration in bovine ISMFs (passage 3 and 7). However, at passage 11, impairment in intracellular Ca2+ responses was observed. Thus, bovine ISMFs might be a novel useful tool with long life span and good cellular responses to investigate physiological/pathophysiological roles of ISMFs.

Bone marrow-derived CX3CR1 progenitors contribute to neointimal smooth muscle cells via fractalkine CX3CR1 interaction.

Smooth muscle cells play a major role in numerous vascular diseases that contribute to remodeling, repair after injury, and arteriogenesis, and the source of these cells is thought to lie within the vessel wall and the circulating blood. Currently, the precise origin and mechanism of differentiation of extravascular smooth muscle progenitor cells (SPCs) is unclear. We show here that the CX(3)CR1 mononuclear cell population of murine bone marrow provides a source of SPCs that contributes to smooth muscle cells within the neointimal plaque after vascular injury. Moreover, CX(3)CR1-fractalkine (FKN) interaction in vivo is essential for smooth muscle cell differentiation of bone marrow-derived progenitor cells at the vessel wall level. Functional competence of bone marrow-derived CX(3)CR1 positive cells to interact with FKN is also crucial in part for neointima formation following vascular injury. Finally, in a pure preparation of bone marrow-derived CX(3)CR1 positive cells, we show that in vitro smooth muscle cell differentiation increases markedly in the presence of FKN. Our data highlight a novel functional relationship between the myeloid and vascular systems and in the context of vascular injury and repair underscores a key chemokine-receptor pathway that may regulate cell fate when smooth muscle cell differentiation is required.

Nkx2.5/Csx represses myofibroblast differentiation.

Myofibroblasts are known to play key roles in wound healing and fibrosis. They are thought to arise de novo from fibroblasts, and are characterized by induction of alpha-smooth muscle actin (alpha-SMA) expression. The homeobox transcription factor Csx/Nkx2.5 is critical in heart development and cardiogenesis by inducing expression of genes associated with cardiomyocyte differentiation. Here, we report a novel repressor activity of Nkx2.5 on myofibroblast differentiation. Because a key marker of myofibroblast differentiation is expression of the alpha-SMA gene, we first scanned its promoter region for possible cis-acting elements. The results show three potential binding sites (designated Nkx2.5 element [NKE]-1, -2, and -3) containing the Nkx2.5/Csx consensus binding motif (5'-TNNAGTG-3'). To determine their functional importance, site-directed mutagenesis directed at these elements individually and in combination indicated that mutation of NKE1 and -3 significantly enhanced alpha-SMA gene promoter activity, whereas mutation of NKE2 did not have a significant effect. The results of gel shift assays confirmed that Nkx2.5 could bind to both NKE1 and -3, but not to NKE2. Consistent with the mutagenesis studies, ectopically induced expression of Nkx2.5 inhibited alpha-SMA gene expression. Analysis of nkx2.5 gene expression indicates that it was significantly induced by basic fibroblast growth factor treatment of isolated lung fibroblasts in vitro. In vivo, lung Nkx2.5 expression was significantly diminished in bleomycin-induced pulmonary fibrosis. These findings are consistent with a novel function for Nkx2.5 as a repressor of alpha-SMA gene transcription, which may be of homeostatic significance as a means of suppressing myofibroblast differentiation in the absence of tissue injury.

The influence of endothelial cells on the ECM composition of 3D engineered cardiovascular constructs.

Tissue engineering of small diameter (<5 mm) blood vessels is a promising approach to develop viable alternatives for autologous vascular grafts. Development of a functional, adherent, shear resisting endothelial cell (EC) layer is one of the major issues limiting the successful application of these tissue engineered grafts. The goal of the present study was to create a confluent EC layer on a rectangular 3D cardiovascular construct using human venous cells and to determine the influence of this layer on the extracellular matrix composition and mechanical properties of the constructs. Rectangular cardiovascular constructs were created by seeding myofibroblasts (MFs) on poly(glycolic acid) poly-4-hydroxybutyrate scaffolds using fibrin gel. After 3 or 4 weeks, ECs were seeded and co-cultured using EGM-2 medium for 2 or 1 week, respectively. A confluent EC layer could be created and maintained for up to 2 weeks. The EGM-2 medium lowered the collagen production by MFs, resulting in weaker constructs, especially in the 2 week cultured constructs. Co-culturing with ECs slightly reduced the collagen content, but had no additional affect on the mechanical performance. A confluent endothelial layer was created on 3D human cardiovascular constructs. The layer was co-cultured for 1 and 2 weeks. Although, the collagen production of the MFs was slightly lowered, co-culturing ECs for 1 week results in constructs with good mechanical properties and a confluent EC layer.

Role of endothelin receptors on basal and endothelin-1-stimulated lung myofibroblast proliferation.

Proliferation of myofibroblasts (MYF) contributes to numerous lung disorders. Endothelin-1 (ET-1) production is increased in various lung diseases and could contribute to lung remodelling. The respective roles of ETA and ETB receptors (ETA-R, ETB-R) and the role of endogenous ET-1 production by lung MYF on proliferation of MYF remain uncertain. Rat lung MYF were isolated and 3H-thymidine and 3H-leucine incorporation assays were completed in the presence of a selective ETA-R antagonist, a selective ETB-R antagonist, or a combination of both. Receptor expression was evaluated by confocal imaging, and ET-1 levels were measured by ELISA. Confocal microscopy revealed abundant ETA-R and ETB-R expression on lung MYF. ET-1 (10 nmol/L) stimulated MYF proliferation and protein synthesis through PI3-kinase and p38 pathways. Although neither selective ETA-R blockade (BQ-123, 1 micromol/L) nor selective ETB-R blockade (BQ-788, 1 micromol/L) alone inhibited proliferation or protein synthesis, their combination almost completely abolished ET-1 mitogenic effect. Surprisingly, basal MYF proliferation was increased by selective blockade of either ETA-R or ETB-R alone, but not by dual blockade. ET-1 levels were not affected by the antagonists. Our findings indicate that both the ETA-R and the ETB-R regulate basal and stimulated lung MYF proliferation and suggest possible interactions between the receptors.

[Effects of interferon-gamma on tubular epithelial-myofibroblast transdifferentiation in vitro].

OBJECTIVE: To investigate the effects of interferon-gamma (IFN-gamma) on tubular epithelial-myofibroblast transdifferentiation (TEMT) induced by transforming growth factor (TGF-beta1). METHODS: The normal rat kidney tubular epithelial cells (NRK52E) were cultured and divided into blank (NRK52E cells only) control group, TGF-beta1 (3 ng/mL) treated group, IFN-gamma (1000 IU/mL) treated group, and IFN-gamma inhibition group (TGF-beta1 3 ng/mL + IFN-gamma 200, 400, 600, 1000, 2000, 3000 IU/mL). After 72 hours of treatment, the morphology of cells was observed under phase-contrast microscopy and scanning electron microscopy. The expressions of a-smooth muscle actin (alpha-SMA) and connective tissue growth factor (CTGF) were detected by immunocytochemistry. Flowcytometry was employed to measure the percentage of alpha-SMA+ cells and the mean channel fluorescence (MCF). The expressions of alpha-SMA mRNA and CTGF mRNA were examined by reverse transcription-polymerase chain reaction analyses (RT-PCR). The level of collagen in the culture supernatant was measured by Enzyme-linked immunoadsordent assay (ELISA). RESULTS: NRK52E cells cultured in the control group showed a classic cobblestone morphology. TGF-beta1 induced NRK52E cells to transdifferentiate into myofibroblast-like cells, which showed strong alpha-SMA immunostaining. The TGF-beta1 treated cells had higher percentage of a-SMA+ cells, MCF and alpha-SMA mRNA, increased CTGF mRNA expression, and ascended collagen III than the blank controls (P<0.05). IFN-gamma treated alone did not make any changes to the cell morphology, the expressions of alpha-SMA mRNA and CTGF mRNA and the level of collagen III (P>0.05). IFN-gamma exerted a strong inhibitory effect on the TEMT induced by TGF-beta1. With the increase of IFN-gamma, the percentage of alpha-SMA+ cells, the level of collagen III, and the expressions of alpha-SMA mRNA and CTGF mRNA decreased (P<0.05). CONCLUSION: IFN-gamma inhibits the TEMT induced by TGF-beta1 and reduces the level of collagen III.

The effect of matrix density on the regulation of 3-D capillary morphogenesis.

The means by which extracellular matrix density regulates three-dimensional capillary morphogenesis is unclear. To study this phenomenon, we utilized a fibrin-based in vitro assay in which a fibroblast monolayer is plated atop a fibrin gel approximately 2.5 mm away from endothelial cell-coated beads within the matrix. Increasing fibrin density from 2.5 to 10 mg/ml resulted in a threefold reduction in capillary network formation. However, distributing fibroblasts throughout the matrix completely eliminated this inhibitory effect, resulting in robustly vascularized matrices suitable for in vivo applications, as functional anastomoses formed between the implanted tissues and host vasculature when implanted into immune-compromised mice. Dense matrices did not stimulate fibroblast-mediated matrix remodeling: differentiation into myofibroblasts, matrix production, and protease secretion were not enhanced by the dense condition. Instead, quantifying diffusivity of FITC-dextran (molecular mass 10, 40, 70, and 150 kDa) through fibrin revealed a two- to threefold decrease within the 10 mg/ml matrices. Thus, distributing a proangiogenic source (fibroblasts) throughout the matrix stimulates capillary network formation by overcoming this diffusion restriction due to significantly reduced diffusion distances. Although roles for matrix stiffness and ligand binding density have previously been identified, our results emphasize the importance of diffusion restrictions in limiting capillary morphogenesis.