Collagen microencapsulation recapitulates mesenchymal condensation and potentiates chondrogenesis of human mesenchymal stem cells - A matrix-driven in vitro model of early skeletogenesis.

Mesenchymal condensation is a critical transitional stage that precedes cartilage or bone formation. A microencapsulation technique was previously established to entrap mesenchymal stem cells (MSC) in nanofibrous collagen meshwork. We hypothesize that collagen microencapsulation of MSCs mimics the mesenchymal cell condensation process. Specifically, human MSCs at different concentrations were microencapsulated in collagen for different time points before evaluation for early skeletogenesis markers. A transient upregulation of mesenchymal condensation markers including peanut agglutinin, fibronectin, integrins α5 and αv, an enhanced nuclear localization of SOX9 and binding interactions with COL2A1, and other changes in chondrogenic, hypertropic and osteogenic marker were demonstrated. Collagen microencapsulation upregulated both the chondrogenic and the osteogenic transcription factors and the encapsulated hMSCs hold the potential to differentiate towards both chondrogenic and osteogenic lineages. We also hypothesize that collagen microencapsulation potentiates MSC chondrogenesis. Particularly, chondrogenic differentiation of hMSCs were induced at different time post-encapsulation before evaluation for chondrogenesis outcomes. Sustained SOX9, ACAN and COL2A1 expression were noted and the timing to induce supplement chondro-inductive factors matters. This study reports an extracellular matrix-based in vitro model of mesenchymal condensation, an early stage in skeletogenesis, contributing to rationalizing development-inspired tissue engineering.

Extracellular Protease Inhibition Alters the Phenotype of Chondrogenically Differentiating Human Mesenchymal Stem Cells (MSCs) in 3D Collagen Microspheres.

Matrix remodeling of cells is highly regulated by proteases and their inhibitors. Nevertheless, how would the chondrogenesis of mesenchymal stem cells (MSCs) be affected, when the balance of the matrix remodeling is disturbed by inhibiting matrix proteases, is incompletely known. Using a previously developed collagen microencapsulation platform, we investigated whether exposing chondrogenically differentiating MSCs to intracellular and extracellular protease inhibitors will affect the extracellular matrix remodeling and hence the outcomes of chondrogenesis. Results showed that inhibition of matrix proteases particularly the extracellular ones favors the phenotype of fibrocartilage rather than hyaline cartilage in chondrogenically differentiating hMSCs by upregulating type I collagen protein deposition and type II collagen gene expression without significantly altering the hypertrophic markers at gene level. This study suggests the potential of manipulating extracellular proteases to alter the outcomes of hMSC chondrogenesis, contributing to future development of differentiation protocols for fibrocartilage tissues for intervertebral disc and meniscus tissue engineering.

Competitive Binding Between Id1 and E2F1 to Cdc20 Regulates E2F1 Degradation and Thymidylate Synthase Expression to Promote Esophageal Cancer Chemoresistance.

PURPOSE: Chemoresistance is a major obstacle in cancer therapy. We found that fluorouracil (5-FU)-resistant esophageal squamous cell carcinoma cell lines, established through exposure to increasing concentrations of 5-FU, showed upregulation of Id1, IGF2, and E2F1. We hypothesized that these genes may play an important role in cancer chemoresistance. EXPERIMENTAL DESIGN: In vitro and in vivo functional assays were performed to study the effects of Id1-E2F1-IGF2 signaling in chemoresistance. Quantitative real-time PCR, Western blotting, immunoprecipitation, chromatin immunoprecipitation, and dual-luciferase reporter assays were used to investigate the molecular mechanisms by which Id1 regulates E2F1 and by which E2F1 regulates IGF2. Clinical specimens, tumor tissue microarray, and Gene Expression Omnibus datasets were used to analyze the correlations between gene expressions and the relationships between expression profiles and patient survival outcomes. RESULTS: Id1 conferred 5-FU chemoresistance through E2F1-dependent induction of thymidylate synthase expression in esophageal cancer cells and tumor xenografts. Mechanistically, Id1 protects E2F1 protein from degradation and increases its expression by binding competitively to Cdc20, whereas E2F1 mediates Id1-induced upregulation of IGF2 by binding directly to the IGF2 promoter and activating its transcription. The expression level of E2F1 was positively correlated with that of Id1 and IGF2 in human cancers. More importantly, concurrent high expression of Id1 and IGF2 was associated with unfavorable patient survival in multiple cancer types. CONCLUSIONS: Our findings define an intricate E2F1-dependent mechanism by which Id1 increases thymidylate synthase and IGF2 expressions to promote cancer chemoresistance. The Id1-E2F1-IGF2 regulatory axis has important implications for cancer prognosis and treatment.

Protease inhibitors enhance extracellular collagen fibril deposition in human mesenchymal stem cells.

INTRODUCTION: Collagen is a widely used naturally occurring biomaterial for scaffolding, whereas mesenchymal stem cells (MSCs) represent a promising cell source in tissue engineering and regenerative medicine. It is generally known that cells are able to remodel their environment by simultaneous degradation of the scaffolds and deposition of newly synthesized extracellular matrix. Nevertheless, the interactions between MSCs and collagen biomaterials are poorly known, and the strategies enhancing the extracellular matrix deposition are yet to be defined. In this study, we aim to investigate the fate of collagen when it is in contact with MSCs and hypothesize that protease inhibition will enhance their extracellular deposition of collagen fibrils. METHODS: Specifically, human MSCs (hMSCs) were exposed to fluorescence-labeled collagen with and without intracellular or extracellular protease inhibitors (or both) before tracing the collagen at both intracellular and extracellular spaces. RESULTS: Collagen were internalized by hMSCs and degraded intracellularly in lysosomes. In the presence of protease inhibitors, both intracellular collagen fibril growth and extracellular deposition of collagen fibrils were enhanced. Moreover, protease inhibitors work synergistically with ascorbic acid, a well-known matrix deposition-enhancing reagent, in further enhancing collagen fibril deposition at the extracellular space. CONCLUSION: These findings provide a better understanding of the interactions between hMSCs and collagen biomaterials and suggest a method to manipulate matrix remodeling and deposition of hMSCs, contributing to better scaffolding for tissue engineering and regenerative medicine.

Mechanoresponsive, omni-directional and local matrix-degrading actin protrusions in human mesenchymal stem cells microencapsulated in a 3D collagen matrix.

Cells are known to respond to multiple niche signals including extracellular matrix and mechanical loading. In others and our own studies, mechanical loading has been shown to induce the formation of cell alignment in 3D collagen matrix with random meshwork, challenging our traditional understanding on the necessity of having aligned substrates as the prerequisite of alignment formation. This motivates our adventure in deciphering the mechanism of loading-induced cell alignment and hence the discovery of the novel protrusive functional structure at the cell-matrix interface. Here we report the formation of mechanoresponsive, omni-directional and local matrix-degrading actin protrusions in human mesenchymal stem cells (hMSCs) microencapsulated in collagen following a shifted actin assembly/disassembly balance. These actin protrusive structures exhibit morphological and compositional similarity to filopodia and invadopodia but differ from them in stability, abundance, signaling and function. Without ruling out the possibility that these structures may comprise special subsets of filopodia and invadopodia, we propose to name them as mechanopodia so as to reveal their mechano-inductive mechanism. We also suggest that more intensive investigations are needed to delineate the functional significance and physiological relevance of these structures. This work identifies a brand new target for cell-matrix interaction and mechanoregulation studies.

Scaffold composition affects cytoskeleton organization, cell-matrix interaction and the cellular fate of human mesenchymal stem cells upon chondrogenic differentiation.

The stem cell niche, or microenvironment, consists of soluble, matrix, cell and mechanical factors that together determine the cellular fates and/or differentiation patterns of stem cells. Collagen and glycosaminoglycans (GAGs) are important scaffolding materials that can mimic the natural matrix niche. Here, we hypothesize that imposing changes in the scaffold composition or, more specifically, incorporating GAGs into the collagen meshwork, will affect the morphology, cytoskeletal organization and integrin expression profiles, and hence the fate of human mesenchymal stem cells (MSCs) upon the induction of differentiation. Using chondrogenesis as an example, we microencapsulated MSCs in three scaffold systems that had varying matrix compositions: collagen alone (C), aminated collagen (AC) and aminated collagen with GAGs (ACG). We then induced the MSCs to differentiate toward a chondrogenic lineage, after which, we characterized the cell viability and morphology, as well as the level of cytoskeletal organization and the integrin expression profile. We also studied the fate of the MSCs by evaluating the major chondrogenic markers at both the gene and protein level. In C, MSC chondrogenesis was successfully induced and MSCs that spread in the scaffolds had a clear actin cytoskeleton; they expressed integrin α2ß1, α5 and αv; promoted sox9 nuclear localization transcription activation; and upregulated the expression of chondrogenic matrix markers. In AC, MSC chondrogenesis was completely inhibited but the scaffold still supported cell survival. The MSCs did not spread and they had no actin cytoskeleton; did not express integrin α2 or αv; they failed to differentiate into chondrogenic lineage cells even on chemical induction; and there was little colocalization or functional interaction between integrin α5 and fibronectin. In ACG, although the MSCs did not express integrin α2, they did express integrin αv and there was strong co-localization and hence functional binding between αv and fibronectin. In addition, vimentin was the dominant cytoskeletal protein in these cells, and the chondrogenic marker genes were expressed but at a much lower level than in the MSCs encapsulated in C alone. This work suggests the importance of controlling the matrix composition as a strategy to manipulate cell-matrix interactions (through changes in the integrin expression profile and cytoskeleton organization), and hence stem cell fates.

Bioengineering a multicomponent spinal motion segment construct--a 3D model for complex tissue engineering.

Intervertebral disc degeneration is an important clinical problem but existing treatments have significant drawbacks. The ability to bioengineer the entire spinal motion segment (SMS) offers hope for better motion preservation strategies but is extremely challenging. Here, fabrication of a multicomponent SMS construct with complex hierarchical organization from mesenchymal stem cells and collagen-based biomaterials, using a module-based integrative approach, is reported. The construct consists of two osteochondral subunits, a nucleus pulposus (NP-)-like core and a multi-lamellae annulus fibrosus (AF-)-like component. Chondrogenic medium is crucial for stabilizing the osteochondral subunits, which are shown to allow passive nutrient diffusion, while cyclic compression is necessary for better fiber matrix organization. Cells adhere, survive, and interact with the NP-like core. Cyclic torsional loading stimulates cell alignment in the AF-like lamellae and the number of lamellae affects the mechanical properties of the construct. This work represents an important milestone in SMS tissue engineering and provides a 3D model for studying tissue maturation and functional remodeling.

Id1-induced IGF-II and its autocrine/endocrine promotion of esophageal cancer progression and chemoresistance--implications for IGF-II and IGF-IR-targeted therapy.

PURPOSE: To investigate the autocrine/endocrine role of Id1-induced insulin-like growth factor-II (IGF-II) in esophageal cancer, and evaluate the potential of IGF-II- and IGF-type I receptor (IGF-IR)-targeted therapies. EXPERIMENTAL DESIGN: Antibody array-based screening was used to identify differentially secreted growth factors from Id1-overexpressing esophageal cancer cells. In vitro and in vivo assays were performed to confirm the induction of IGF-II by Id1, and to study the autocrine and endocrine effects of IGF-II in promoting esophageal cancer progression. Human esophageal cancer tissue microarray was analyzed for overexpression of IGF-II and its correlation with that of Id1 and phosphorylated AKT (p-AKT). The efficacy of intratumorally injected IGF-II antibody and intraperitoneally injected cixutumumab (fully human monoclonal IGF-IR antibody) was evaluated using in vivo tumor xenograft and experimental metastasis models. RESULTS: Id1 overexpression induced IGF-II secretion, which promoted cancer cell proliferation, survival, and invasion by activating AKT in an autocrine manner. Overexpression of IGF-II was found in 21 of 35 (60%) esophageal cancer tissues and was associated with upregulation of Id1 and p-AKT. IGF-II secreted by Id1-overexpressing esophageal cancer xenograft could instigate the growth of distant esophageal tumors, as well as promote metastasis of circulating cancer cells. Targeting IGF-II and IGF-IR had significant suppressive effects on tumor growth and metastasis in mice. Cixutumumab treatment enhanced the chemosensitivity of tumor xenografts to fluorouracil and cisplatin. CONCLUSIONS: The Id1-IGF-II-IGF-IR-AKT signaling cascade plays an important role in esophageal cancer progression. Blockade of IGF-II/IGF-IR signaling has therapeutic potential in the management of esophageal cancer.

Delivering mesenchymal stem cells in collagen microsphere carriers to rabbit degenerative disc: reduced risk of osteophyte formation.

Mesenchymal stem cells (MSCs) have the potential to treat early intervertebral disc (IVD) degeneration. However, during intradiscal injection, the vast majority of cells leaked out even in the presence of hydrogel carrier. Recent evidence suggests that annulus puncture is associated with cell leakage and contributes to osteophyte formation, an undesirable side effect. This suggests the significance of developing appropriate carriers for intradiscal delivery of MSCs. We previously developed a collagen microencapsulation platform, which entraps MSCs in a solid microsphere consisting of collagen nanofiber meshwork. These solid yet porous microspheres support MSC attachment, survival, proliferation, migration, differentiation, and matrix remodeling. Here we hypothesize that intradiscal injection of MSCs in collagen microspheres will outperform that of MSCs in saline in terms of better functional outcomes and reduced side effects. Specifically, we induced disc degeneration in rabbits and then intradiscally injected autologous MSCs, either packaged within collagen microspheres or directly suspended in saline, into different disc levels. Functional outcomes including hydration index and disc height were monitored regularly until 6 months. Upon sacrifice, the involved discs were harvested for histological, biochemical, and biomechanical evaluations. MSCs in collagen microspheres showed advantage over MSCs in saline in better maintaining the dynamic mechanical behavior but similar performance in hydration and disc height maintenance and matrix composition. More importantly, upon examination of gross appearance, radiograph, and histology of IVD, delivering MSCs in collagen microspheres significantly reduced the risk of osteophyte formation as compared to that in saline. This work demonstrates the significance of using cell carriers during intradiscal injection of MSCs in treating disc degeneration.

Effects of nucleus pulposus cell-derived acellular matrix on the differentiation of mesenchymal stem cells.

Recent attempts to treat disc degeneration with mesenchymal stem cells (MSCs) showed encouraging results. Differentiating MSCs towards nucleus pulposus cell (NPC)-like lineages represents a speculative mechanism. Niche factors including hypoxia, growth factors and cell-cell interactions have been suggested but the matrix niche factor has not been studied. Our collagen microencapsulation provides a 3D model to study matrix niche as it enables the encapsulated cells to remodel the template matrix. We previously demonstrated the chondro-inductive role of of chondrocytes-derived matrix in MSCs and showed that NPCs maintained their phenotype and remodeled the template matrix of collagen microspheres into a glycosaminoglycan (GAG)-rich one. Here we aim to study the effects of NPC-derived matrix on MSC differentiation towards NPC-like lineages by firstly producing an NPC-derived matrix in collagen microspheres, secondly optimizing a decellularization protocol to discard NPCs yet retaining the matrix, thirdly repopulating the acellular NPC-derived matrix with MSCs and fourthly evaluating their phenotype. Finally, we injected these microspheres in a pilot rabbit disc degeneration model. Results showed that NPCs survived, maintained their phenotypic markers and produced GAGs. A decellularization protocol with maximal removal of the NPCs, minimal loss in major matrix components and partial retention of NPC-specific markers was identified. The resulting acellular matrix supported MSC survival and matrix production, and up-regulated the gene expression of NPC markers including type II collagen and glypican 3. Finally, injection of MSC in these microspheres in rabbit degenerative disc better maintained hydration level with more pronounced staining of GAGs and type II collagen than controls.

Targeting NF-kappaB signaling pathway suppresses tumor growth, angiogenesis, and metastasis of human esophageal cancer.

Esophageal cancer is the eighth most common malignancy, and one of the leading causes of cancer-related deaths worldwide. The overall 5-year survival rate of patients with esophageal cancer remains low at 10% to 40% due to late diagnosis, metastasis, and resistance of the tumor to radiotherapy and chemotherapy. NF-kappaB is involved in the regulation of cell growth, survival, and motility, but little is known about the role of this signaling pathway in the tumorigenesis of human esophageal squamous cell carcinoma (ESCC), the most common form of esophageal cancer. This study aims to explore the functions of NF-kappaB in human ESCC progression and to determine whether targeting the NF-kappaB signaling pathway might be of therapeutic value against ESCC. Our results from human ESCC cell lines and ESCC tissue indicated that NF-kappaB is constitutively active in ESCC. Exposure of ESCC cells to two NF-kappaB inhibitors, Bay11-7082 and sulfasalazine, not only reduced cancer cell proliferation, but also induced apoptosis and enhanced sensitivity to chemotherapeutic drugs, 5-fluorouracil, and cisplatin. In addition, Bay11-7082 and sulfasalazine suppressed the migration and invasive potential of ESCC cells. More importantly, the results from tumor xenograft and experimental metastasis models showed that Bay11-7082 had significant antitumor effects on ESCC xenografts in nude mice by promoting apoptosis, and inhibiting proliferation and angiogenesis, as well as reduced the metastasis of ESCC cells to the lungs without significant toxic effects. In summary, our data suggest that NF-kappaB inhibitors may be potentially useful as therapeutic agents for patients with esophageal cancer.

Id-1 promotes tumorigenicity and metastasis of human esophageal cancer cells through activation of PI3K/AKT signaling pathway.

Id-1 (inhibitor of differentiation or DNA binding) is a helix-loop-helix protein that is overexpressed in many types of cancer including esophageal squamous cell carcinoma (ESCC). We previously reported that ectopic Id-1 expression activates the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) signaling pathway in human esophageal cancer cells. In this study, we confirmed a positive correlation between Id-1 and phospho-AKT (Ser473) expressions in ESCC cell lines, as well as in ESCC on a tissue microarray. To investigate the significance of Id-1 in esophageal cancer progression, ESCC cells with stable ectopic Id-1 expression were inoculated subcutaneously into the flank of nude mice and were found to form larger tumors that showed elevated Ki-67 proliferation index and increased angiogenesis, as well as reduced apoptosis, compared with control cells expressing the empty vector.The Id-1-overexpressing cells also exhibited enhanced metastatic potential in the experimental metastasis assay. Treatment with the PI3K inhibitor LY294002 attenuated the tumor promotion effects of Id-1, indicating that the effects were mediated by the PI3K/AKT signaling pathway. In addition, our in vitro experiments showed that ectopic Id-1 expression altered the expression levels of markers associated with epithelial-mesenchymal transition and enhanced the migration ability of esophageal cancer cells. The Id-1-overexpressing ESCC cells also exhibited increased invasive potential, which was in part due to PI3K/AKT-dependent modulation of matrix metalloproteinase-9 expression. In conclusion, our results provide the first evidence that Id-1 promotes tumorigenicity and metastasis of human esophageal cancer in vivo and that the PI3K inhibitor LY294002 can attenuate these effects.

Coexpression of adrenomedullin and its receptors in the reproductive system of the rat: effects on steroid secretion in rat ovary.

The present study demonstrates the expression of adrenomedullin (ADM) in the reproductive system of the female rat and its effect on the secretion of estradiol and progesterone. Ovarian ADM and Adm mRNA levels were decreased at estrus, whereas oviductal Adm mRNA levels were low at proestrus. Both tissues were shown to coexpress mRNAs encoding the calcitonin receptor-like receptor and receptor activity-modifying protein 1 (Ramp1), Ramp2, and Ramp3. Gel filtration chromatography of ovarian extracts showed two peaks, with the predominant one eluting at the position of authentic rat ADM (1-50) at estrus and at the position of ADM precursor at diestrus. Positive ADM immunostaining was localized in the granulosa and theca cells of the follicle and corpora lutea of the ovary. Adrenomedullin inhibited FSH-induced estradiol secretion in 2-day-old follicles and also suppressed eCG-stimulated progesterone release in corpora lutea. The inhibitory effect of ADM on the follicles and the corpora lutea was abolished by calcitonin gene-related peptide (8-37) and ADM (22-52), respectively. The presence of ADM and the gene expression of ADM and its receptor components in the female reproductive system suggest a paracrine effect of ADM on ovarian steroidogenesis.

Differential regulation of adrenomedullin gene expression in the fundic and pyloric regions of the rat stomach during acute and chronic starvation.

Adrenomedullin (AM) has been shown to be present in the stomach but the role of gastric AM is obscure. To investigate the effects of starvation on AM in the stomach, we studied the changes in gene expression of preproadrenomedullin (preproAM) and AM receptors by reverse transcription-polymerase chain reaction (RT-PCR), and tissue AM concentrations by radioimmunoassay (RIA) in the fundus and pylorus of the stomach of rats subjected to either acute (1-day) or chronic (4-day) starvation. An up-regulation of preproAM gene expression was observed in the fundus after acute starvation, and in the pylorus after chronic starvation. Immunoreactive AM (ir-AM) levels were increased in both fundus and pylorus after chronic starvation. In addition, marked reductions in the gene expression of fundic calcitonin receptor-like receptor (CRLR) and receptor activity-modifying protein (RAMP) 3 as well as the pyloric CRLR and RAMP2 were observed in the chronically starved rats. The present study suggests that the gene expression of preproadrenomedullin mRNA is differentially regulated by starvation in the different parts of the stomach.

Effect of aging on the expression of adrenomedullin and its receptor component proteins in the male reproductive system of the rat.

This study investigated the levels of adrenomedullin (AM) and the gene expression of AM, calcitonin receptor-like receptor (CRLR), receptor activity-modifying proteins (RAMPs), and receptor-coupling protein (RCP) in the testis, ventral prostate, seminal vesicle, and epididymis in rats aged 3, 12, and 20 months by radioimmunoassay and semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). The results indicate an age-related increase in AM and its messenger RNA (mRNA) levels in the testis and decrease in the sex accessory glands. The gene expression of CRLR and RCP decreased only in the sex accessory glands. The changes in the gene expression of RAMPs suggest that the major increase was in CGRP receptors in the testis, whereas the major decreases in the ventral prostate and the seminal vesicles might be CGRP and AM receptors, respectively. The decreases in these receptors were similar in the epididymis. The results suggest possible roles of AM in the male reproductive system during aging.

Adrenomedullin peptide: gene expression of adrenomedullin, its receptors and receptor activity modifying proteins, and receptor binding in rat testis--actions on testosterone secretion.

Adrenomedullin (ADM) has been shown to be present in the human and rat male reproductive systems. This study demonstrates the expression of ADM in the rat testis and its effect on the secretion of testosterone. Whole testicular extracts had 5.43 +/- 0.42 fmol of immunoreactive ADM per milligram of protein and 84 +/- 8 fg of ADM mRNA per picogram of Actb (beta-actin) mRNA. Immunocytochemical studies showed positive ADM immunostaining in the Leydig cells and in the Sertoli cells. Gel filtration chromatography of testicular extracts showed two peaks, with the predominant one eluting at the position of the ADM precursor. Furthermore, the testis was shown to coexpress mRNAs encoding the calcitonin receptor-like receptor and receptor activity modifying protein 1 (Ramp1), Ramp2, and Ramp3. These account for the specific binding of ADM to the testis, which was partially inhibited by human ADM (22-52) and by human calcitonin gene-related peptide (8-37), the ADM and calcitonin gene-related peptide receptor antagonists, respectively. Administration of ADM to testicular blocks in vitro resulted in a dose-dependent inhibition of hCG-stimulated release of testosterone, which was abolished by the administration of ADM (22-52). Our results suggest a paracrine effect of ADM on testicular steroidogenesis.

The gene expression of adrenomedullin, calcitonin-receptor-like receptor and receptor activity modifying proteins (RAMPs) in CCl4-induced rat liver cirrhosis.

This study was undertaken to determine AM expression in carbon tetrachloride (CCl4)-induced liver cirrhosis developed with peritoneal ascites. Sprague-Dawley rats received subcutaneous injections of CCl4 twice weekly in olive oil (1:1, 0.3 ml per kg body weight) for 6 or 12 weeks until ascites developed, or saline in olive oil as control. At 6 weeks, fibrosis developed and at 12 weeks cirrhosis developed with ascites formation. At both 6 and 12 weeks, increases in plasma renin and AM were evident, as was the gene expression of AM. At 12 weeks after CCl4 injection, the gene expression of calcitonin-like-receptor (CRLR) and receptor activity modifying proteins (RAMP1, RAMP2 and RAMP3) were all elevated when compared to the control. The results suggest that liver cirrhosis increases mRNA expressions of AM, CRLR and RAMP1, RAMP2 and RAMP3 and that the increase in AM gene expression precedes the development of cirrhosis. The increase in AM synthesis as reflected by an increase in AM gene expression, together with a lack of increase in AM peptide at both 6 and 12 weeks may suggest an elevation of AM release. Given the potent vasodilatory action of AM, the increase in the synthesis and release of AM in the cirrhotic liver may also contribute to peripheral vasodilatation in liver cirrhosis.

Adrenomedullin gene expression and levels in the cardiovascular system after treatment with lipopolysaccharide.

To study the effect of septicaemia, the temporal changes in tissue adrenomedullin (AM) and preproAM mRNA levels were studied in the heart and blood vessels after lipopolysaccharide (LPS) injection. Radioimmunoassay and solution hybridization-RNase protection assays were used to follow the changes in AM and its mRNA levels respectively after intraperitoneal injection of 10 mg/kg LPS in rats. The preproAM mRNA levels increased at 1 h in the right atrium after LPS injection, while the AM contents decreased at 1 h in the left atrium. The preproAM mRNA levels increased at 3 and 6 h in the left ventricle, whereas it increased at 6 h in the right ventricles after LPS injection. There was an increase in preproAM mRNA levels at 1 and 3 h in the mesenteric artery, while AM levels were increased at 1, 3 and 6 h. However, there were no such changes in the thoracic aorta. There were also increases in tumour necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta and IL-6 in the heart, and in the mesenteric artery (TNF-alpha and IL-1beta) and in thoracic aorta (IL-1beta and IL-6). The present results suggest that the biosynthesis and secretion of AM may be increased in cardiovascular tissues of rats injected with LPS, and that AM may play multiple roles in inflammation.

Differential induction of adrenomedullin, interleukins and tumour necrosis factor-alpha by lipopolysaccharide in rat tissues in vivo.

The aim of the present study was to determine the temporal changes in tissue adrenomedullin (AM) and cytokine contents and cytokine and preproAM mRNA levels in the kidney, liver, adrenal gland and spleen of lipopolysaccharide (LPS)-treated rats. Rats were injected with LPS (10 mg/kg, i.p.). Radioimmunoassay and solution hybridization-RNase protection assays were used to follow the changes in AM and its mRNA levels, respectively; ELISA and reverse transcription-polymerase chain reaction were used to follow the changes in cytokines and their mRNA levels, respectively. In the kidney, the preproAM mRNA levels were increased 1 and 3 h after LPS treatment, whereas AM levels were decreased at 3 h. Interleukin (IL)-6 and IL-1beta levels were increased at 3 and 6 h, respectively. The preproAM mRNA levels were elevated in the liver 3 h after LPS injection. Concentrations of tumour necrosis factor (TNF)-alpha and IL-1beta were increased at l and 6 h, respectively. There were no changes in the levels of either preproAM mRNA or AM in the adrenal gland and the spleen. In the spleen, TNF-alpha levels were elevated at 1 and 3 h after LPS injection and IL-1beta was elevated at 1 and 6 h after LPS injection, whereas in the adrenal gland IL-1beta was elevated at 6 h after injection. The mRNA levels of the three cytokines were elevated at all three time intervals examined in the kidney, liver, adrenal gland and spleen, with the exception that TNF-alpha mRNA was not elevated in the adrenal gland at 6 h after LPS injection and IL-1beta mRNA was not elevated in the spleen at 3 and 6 h. The plasma concentrations of TNF-alpha were increased at 1 and 3 h after LPS injection, whereas plasma concentration of IL-1beta and IL-6 were elevated at 3 and 6 h for both. The present results suggest that the biosynthesis and secretion of AM may be differentially regulated in various tissues of rats injected with LPS and that AM may interact with cytokines during inflammation.

Adrenomedullin is both proinflammatory and antiinflammatory: its effects on gene expression and secretion of cytokines and macrophage migration inhibitory factor in NR8383 macrophage cell line.

Adrenomedullin (ADM) is a potent vasorelaxant peptide that plays important roles in cardiovascular homeostasis and inflammatory response. ADM derived from macrophages is one of the major sources of ADM that is produced in the inflammatory process. To assess the functions of ADM in inflammation, we studied the temporal changes in ADM production and its effect on secretion of macrophage migration inhibitory factor (MIF) and cytokine response of NR8383 rat macrophages activated by lipopolysaccharide (LPS). NR8383 cells were stimulated by LPS in the absence and presence of exogenous ADM, and the concentrations of ADM, MIF, and proinflammatory cytokines (IL-6, TNF-alpha, and IL-1beta) in the culture media and gene expressions of the cells were measured. We confirmed that the secretion and mRNA expression of ADM in the macrophages were markedly increased by LPS. ADM increased initial secretion of MIF and IL-1beta from both nonstimulated and LPS-stimulated cells, and it also increased basal and LPS-induced IL-6 secretion of the cells by 2- to 15-fold. However, it reduced secretion of TNF-alpha from LPS-stimulated cells by 34-56%. Our results suggest that ADM modulates MIF secretion and cytokine production and plays important roles in both the initiation and propagation of the inflammatory response.