Increased epithelial cell proliferation and abnormal extracellular matrix in rat polycystic kidney disease

Proliferation of renal tubular epithelial cells is considered a major factor leading to cyst formation in human polycystic kidney disease (PKD). The Han:SPRD rat model for inherited PKD permits a close scrutiny, especially for early stages of the disease, and shows numerous similarities to human autosomal dominant PKD (ADPKD). In this study, the exact in vivo proliferation rate in Han:SPRD rat kidneys was evaluated in a cell type-specific manner, using immunohistochemistry with antibody to proliferating cell nuclear antigen (PCNA). The proliferation index (PI; percentage of PCNA-positive cell nuclei) was determined in normal and cystically altered tissue, and a relationship between proliferative activity and alterations in extracellular matrix expression was established using in situ hybridization for collagen I and IV mRNA. Heterozygously affected rats (cy/+) showed strong increases of PI values in cystically altered nephron portions that were mostly derived from proximal tubule. Cell proliferation obviously preceded cyst formation, because early in the progression of the disease, the normal-appearing tubules from PKD kidneys had markedly increased PI values compared with healthy controls (14.1-fold in 3-mo-old rats and 11.9-fold in 12-mo-old rats; P < 0.05), whereas later stages revealed a more generalized cystic degeneration of the nephron, with increases in PI between 14- and 82-fold, depending on the respective category of cystic epithelia. In cysts with a distal phenotype, changes were less pronounced. No significant differences were encountered between the two age groups. Proliferation was also present in interstitial cells, whereas glomeruli were unchanged. Increases in epithelial and interstitial proliferation coincided with an overexpression of matrix compounds. For comparison, changes in homozygously affected rats (cy/cy) showed up to several hundred-fold elevated PI values. These results indicate that in the Han:SPRD model for ADPKD, cystic malformation of the nephron is preceded by and coincides with enhanced epithelial and interstitial cell proliferation. Altered cell-matrix interactions seem to be directly involved in the disruption of epithelial differentiation.     

A macrophage-dependent factor that stimulates the proliferation of fibroblasts in vitro

Whole blood serum (HBS) stimulates the proliferation of fibroblasts in vitro, while platelet-poor plasma serum (PPPS) does not. Fibroblasts grown in the presence of PPPS are truly quiescent in that they are not deprived of nutrients in the culture medium and less than 3% of the cells synthesize DNA and divide. In vivo experiments have suggested that macrophages are necessary for stimulation of fibroplasia during wound repair. We have utilized the difference in growth-promoting activity between HBS and PPPS to study the ability of macrophages to produce growth-promoting activity in cell culture. Guinea pig peritoneal macrophages cultured in vitro in medium containing PPPS release into the medium, either directly or indirectly, a factor (or factors) that stimulates the proliferation of guinea pig wound fibroblasts. This macrophage-dependent, fibroblast-stimulating activity (MFSA) is nondialyzable, heat stable (56 C for 30 minutes), and requires culture in vitro for demonstration of activity. The relationship between MFSA and other growth factor(s) has not yet been determined. In contrast to the macrophage, lymphocytes prepared from mesenteric lymph nodes produced no figroblast-stimulating activity.     

Boron modulates extracellular matrix and TNF alpha synthesis in human fibroblasts

Boric acid was not mitogenic for human fibroblasts and it did not change cell viability until 0.5% (w/v). Boric acid treatment affected the metabolism of human dermal fibroblasts in culture, decreasing the synthesis of extracellular matrix macromolecules such as proteoglycans, collagen, and total proteins. It also increased the release of these molecules into the culture medium. The principal proteins secreted into the medium after boric acid treatment had molecular masses of 90, 70, 58, 49, and 43 kDa and faint bands were detected by electrophoresis between 14 and 30 kDa. hsp 70 and TNF alpha were detected among the secreted proteins by immunoblotting, and the amount of TNF alpha released was quantified by radioimmunoassay. Total mRNA levels were higher after boric acid treatment and peaked after 6 h of treatment. TNF alpha mRNA was undetectable in unstimulated fibroblasts and two TNF alpha mRNA bands were detected after stimulation: immature mRNA (4.8 kb) and mature TNF alpha mRNA (1.9 kb). Thus, the effects of boric acid observed in wound repair in vivo may be due to TNF alpha synthesis and secretion.     

Different human tenascin-C variants in the extracellular matrix of cultured human fibroblasts

Using an immunoadsorbent prepared with a monoclonal antibody specific for the high molecular mass isoform of human tenascin-C, we purified tenascin-C molecules containing at least one large subunit from the extracellular matrix of cultured normal human fibroblasts. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunoblotting analyses have shown that both high and low molecular mass subunits are present in these tenascin-C preparations. Because the monoclonal antibody used is able to bind only the high molecular mass isoform, the present data show that part of the tenascin-C present in the fibroblast extracellular matrix is made up of heterohexameric molecules.     

Tumor necrosis factor-alpha alters steroidogenesis and stimulates proliferation of human ovarian granulosal cells in vitro

OBJECTIVE: To determine if tumor necrosis factor alpha (TNF-alpha) altered human granulosa-luteal cell proliferation and steroidogenesis. DESIGN: Aspirates of follicles from women undergoing in vitro fertilization were subjected to Percoll gradients to collect an enriched population of granulosa-luteal cells. The granulosa-luteal cells were subjected to culture for a period of 10 or 20 days in the presence or absence of various doses of human recombinant TNF-alpha (0.1 to 10.0 ng/mL). PATIENTS: Granulosa-luteal cells from nine patients were evaluated for their response to TNF-alpha in vitro. Patients with three follicles > 16 mm and a serum estradiol (E2) concentration of > 1,836 pmol/L were selected for study. RESULTS: Tumor necrosis factor-alpha increased granulosa-luteal cell number. By day 10 of culture, 10 ng TNF-alpha/mL doubled cell number and > 95% of the cells exhibited 3 beta-hydroxysteroid dehydrogenase. Tumor necrosis factor-alpha at 10 ng/mL increased progesterone (P) accumulation from day 4 through day 20 of culture. Tumor necrosis factor-alpha also increased E2 secretion but in a biphasic manner. During the first 14 days of culture, TNF-alpha increased E2, but thereafter E2 decreased to basal values by day 20. When steroidogenesis was expressed per 1,000 cells per days of culture, TNF-alpha did not increase P beyond controls but significantly increased E2 for the first 14 days of culture after which E2 per 1,000 cells declined. CONCLUSIONS: The results indicate that TNF-alpha stimulates granulosal-luteal cell growth and E2 secretion in vitro, and thus TNF-alpha may promote cellular events associated with formation of the corpus luteum; i.e., granulosa-cell proliferation and steroidogenesis.     

The effect of the extracellular matrix on the adhesion and proliferation of epidermocytes in culture

The influence of types I, III collagen and fibronectin on cultured epidermocytes was studied. The extracellular matrix in vitro stimulated not only adhesion, but also cell proliferation. The highest degree of epidermocytic proliferation on fibronectin matrix was observed.     

Inhibition of glomerular mesangial cell proliferation and extracellular matrix deposition by halofuginone

Mesangial cell proliferation, increased deposition of collagen, and expansion of the mesangial extracellular matrix (ECM) are key features in the development of mesangioproliferative diseases. Halofuginone, a low molecular weight anti-coccidial quinoazolinone derivative, inhibits collagen type alpha 1(I) gene expression and synthesis. We investigated the effect of halofuginone on both normal and SV40 transformed mesangial cell proliferation, collagen synthesis, and ECM deposition. Proliferation of both cell types was almost completely inhibited in the presence of 50 ng/ml halofuginone. The cells were arrested in the late G1 phase of the cell cycle and resumed their normal growth rate following removal of the compound from the culture medium. The antiproliferative effect of halofuginone was associated with inhibition of tyrosine phosphorylation of cellular proteins. Similar results were obtained whether the mesangial cells were seeded on regular tissue culture plastic or in close contact with a naturally produced ECM resembling their local environment in vivo. Halofuginone also inhibited synthesis and deposition of ECM by mesangial cells as indicated by a substantial reduction in 14C-glycine and Na2(35)SO4 incorporation into the ECM, and by the inhibition of collagen type I synthesis and gene expression. It is proposed that by inhibiting collagen type I synthesis and matrix deposition, halofuginone exerts a potent antiproliferative effect that may be applied to inhibit mesangial cell proliferation and matrix expansion in a variety of chronic progressive glomerular diseases.     

UVB irradiation alters cellular responses to cytokines: role in extracellular matrix gene expression

Chronic myeloid leukemia course was evaluated versus sex in 271 patients. Chronic stage involved more pronounced leukocytosis, thrombocytosis and splenomegaly in females, the latter showing higher susceptibility to anemia. As a result, treatment has to deal with a greater mass of tumor. A relatively longer survival time in males (44 and 42 months, respectively) suggest a higher effectiveness of therapy in such patients. When diagnosed, leukocytosis, thrombocytosis, enhanced splenomegaly and anemia should be regarded as factors of unfavorable prognosis.     

Neonatal chorda tympani transection alters adult preference for ammonium chloride in the rat.

The immature gustatory system of the neonatal rat is characterized by sensitivity to disruption by early interventions such as receptor or nerve damage. The present studies examined the effect of chorda tympani transection (neoCTX) of neonates on adult preference for salt and nonsalt stimuli. NeoCTX at 10 days of age led to a striking change in adult rats' preference for NH?C1 solutions but little change in preference for other solutions, including NaC1 and KC1. Permanent anatomical effects of neoCTX included failure of the nerve to regenerate and a loss of all fungiform taste buds. Preference for NH?C1 was not due to an inability to discriminate it from NaC1. Following taste aversion conditioning to NaC1, neoCTX rats clearly distinguished between NaC1 and NH?C1. The effects on NH?C1 preference reflect a sensitive period during development because adult rats receiving similar surgery did not show any change in NH?C1 preference. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Milking procedure alters the electrolyte composition of spontaneously hypertensive and normotensive rat milk

A number of groups, including our own, have shown that the severity of hypertension expressed by the adult spontaneously hypertensive rat (SHR), which is primarily considered to be a genetic model of hypertension, can be reduced as a result of exposure to the behavioural and nutritional environment provided by a normotensive foster mother. It has been suggested that the hypertensive influence of the SHR dam may involve increased sodium delivery to the pups and there have been some reports of elevated sodium concentrations in the milk of SHR dams. However, these studies used either a long (> or =6 h) dam-pup separation period before collecting milk or repeated milking of the same dams, both of which have been shown to alter the trace element content of rat milk. Therefore, we have compared the electrolyte content of milk collected by these methods with milk derived from SHR and normotensive Wistar-Kyoto (WKY) dams separated from their litters for 2 h prior to a single-milking session. Long separation and repeated milking resulted in variable effects on the electrolyte content of both SHR and WKY dams' milk, compared with milk collected after 2 h from dams which had not previously been milked. The most notable effects were the abolition of significant strain differences, observed following 2-h separation, for milk sodium (WKY 22.1+/-1.4 vs. SHR 27.5+/-2.1 mmol/liter, p < 0.05) and calcium (WKY 92.3+/-4.3 vs. SHR 69.4+/-2.9 mmol/liter, p < 0.05) when dams were separated for 6 h or were serially milked. These data suggest that the electrolyte content of SHR and WKY dams' milk can be altered by the collection procedure and it is recommended that dams be milked on only one occasion following a short separation period from their litter.     

Smooth muscle cell diversity and the extracellular matrix in a rat model of restenosis

BACKGROUND: The aortic flap above the facing commissure after removal of the coronary button from the aortic sinus can be utilized for reconstruction in an arterial switch operation. METHODS: The free flap was used to reconstruct the coronary artery in two cases, as a medially based trapdoor. A rotational flap was inserted into the cut-open right pulmonary artery to augment the neopulmonary arterial anastomotic site in another case. RESULTS: All patients are doing well at mid term follow-up. CONCLUSIONS: The indications of each method and reported tricks to avoid coronary artery kinking during an arterial switch operation are discussed.     

Neonatal androgen and estrogen treatments masculinize the size of motoneurons in the rat spinal nucleus of the bulbocavernosus

Electrically-assisted transdermal delivery (EATDD) is the facilitated transport of compounds across the skin using an electromotive force. It has been extensively explored as a potential means for delivering peptides and other hydrophilic, acid-labile or orally unstable products of biotechnology. The predominant mechanism for delivery is iontophoresis, although electroosmosis and electroporation have also been investigated. The focus of this review is to put these different mechanisms in perspective and relate them to the drug and skin model system being investigated.     

Nitric oxide modulates the synthesis of extracellular matrix proteins in cultured rat mesangial cells

Nitric oxide (NO) is an important effector molecule of the inflammatory response. It is synthesized by mesangial cells and has been proposed to contribute to glomerular injury in various disease states. We studied whether NO modulates extracellular matrix production in cultured rat mesangial cells. Stimulation of rat mesangial cell NO release with gamma-interferon and lipopolysaccharide resulted in reduced production of collagen (by 35%) fibronectin (by 48%) (P < 0.05). In contrast, laminin synthesis was enhanced two-fold by the same maneuver (P < 0.05). These changes were reversed by the addition of L-NAME, a selective inhibitor of inducible nitric oxide synthase. This is the first demonstration that NO regulates the synthesis of extracellular matrix by mesangial cells. The results indicate that increased renal production of NO in glomerular diseases may attenuate the production and accumulation of matrix proteins and limit the severity of glomerulosclerosis.     

Function of proteoglycans in the extracellular matrix

Estimating phylogenies from DNA sequence data has become the major methodology of molecular phylogenetics. To date, molecular phylogenetics of the vertebrates has been very dependent on mtDNA, but studies involving mtDNA are limited because the several genes comprising the mt-genome are inherited as a single linkage group. The only apparent solution to this problem is to sequence additional genes, each representing a distinct linkage group, so that the resultant gene trees provide independent estimates of the species tree. There exists the need to find novel gene sequences which contain enough phylogenetic information to resolve relationships between closely related species. A possible source is the nuclear-encoded introns, because they evolve more rapidly than exons. We designed primers to amplify and sequence the 7 intron from the beta-fibrinogen gene for a recently evolved group, the woodpeckers. We sequenced the entire intron for 10 specimens representing five species. Nucleotide substitutions are randomly distributed along the length of the intron, suggesting selective neutrality. A preliminary analysis indicates that the phylogenetic signal in the intron is as strong as that in the mitochondrial encoded cytochrome b (cyt b) gene. The topology of the beta-fibrinogen tree is identical to that of the cyt b tree. This analysis demonstrates the ability of the 7 intron of beta-fibrinogen to provide well resolved, independent gene trees for recently evolved groups and establishes it as a source of sequences to be used in other phylogenetic studies.     

Spatiotemporal patterns of expression of extracellular matrix molecules in the developing and adult rat olfactory system

Using immunocytochemical methods, we have examined extensively the spatial and temporal patterns of expression of three extracellular matrix molecules-laminin, fibronectin, and type IV collagen-in the embryonic, postnatal (days 2 and 11) and adult rat olfactory system. The study started at embryonic day 14 when olfactory fibres and their associated migrating cells course through the nasal mesenchyme. From embryonic day 14 to the adult, a sheet-like pattern of labelling for laminin, fibronectin and type IV collagen was observed along the basal surface of the olfactory epithelium and around the telencephalon. This type of labelling was continuous around the telencephalic vesicle, whereas it appeared disrupted in the basal lamina of the olfactory epithelium to permit exit of the olfactory axons and their associated migrating cells into the mesenchyme. From embryonic day 14 to day 20, punctate labelling for the three molecules studied was observed along the mesenchymal olfactory pathway, the ventral part of the olfactory bulb, the olfactory nerve layer and the presumptive glomerular layer, respectively. By embryonic day 17, the punctate labelling initially detected in the mesenchymal olfactory pathway was replaced by a sheet-like pattern related to the mature basal lamina surrounding the olfactory axon fascicles. Punctate labelling for laminin and type IV collagen persisted in the olfactory nerve layer and around the glomeruli through adult life whereas that of fibronectin declined and disappeared by postnatal day 2. The spatiotemporal distribution of the punctate pattern for laminin, fibronectin and type IV collagen observed in the embryonic olfactory system suggests a role in delineating the pathway for olfactory axon elongation. The continuous expression of laminin and type IV collagen in the adult olfactory bulb may be related to the regenerative activity and high plasticity of the olfactory system.     

Retinoic acid induces changes in the rhombencephalic neural crest cells migration and extracellular matrix composition in chick embryos

Studies on haemopoietic stem cells had led to the realisation that negative feedback inhibitors play an important role in regulating their proliferation. One such molecule was identified as MIP-1 alpha. One of a family of cytokines, originally recognised as inflammatory molecules, MIP-1 alpha is now potentially valuable as a means of manipulating and protecting haemopoietic (and possibly other) stem cells during chemotherapy. This short review briefly considers the structural classification of MIP-1 alpha and its molecular relatives and indicates some of the probable human/murine equivalent molecules outlining the evidence for the equivalence of MIP-1 alpha (murine) and LD78 (human). Sources of MIP-1 alpha/LD78 are identified as monocyte/macrophage and lymphocytic cells and their role in inflammatory responses is seen to be significant. All proliferation in haemopoietic tissue is now recognised as a major target for MIP-1 alpha action. In vitro it synergises with certain growth factors to promote progenitor cell colony formation, but effects are dependent on the maturational age of the cells promoted. With more primitive cells it is seen as inhibitory. This property is particularly valuable in vivo where MIP-1 alpha can protect stem cells against the effects of cytotoxic agents. Since it appears that leukaemic stem cell proliferation is not inhibited, MIP-1 alpha/LD78 present great potential for stem cell protection in the theatre of cytotoxic therapies.     

Fibrolamellar carcinoma of the liver: composition of the extracellular matrix and expression of cell-matrix and cell-cell adhesion molecules

We have analyzed the composition of the tumor stroma and the expression of cell-matrix and cell-cell adhesion molecules in 11 cases of fibrolamellar carcinoma of the liver (FLC), in comparison with 34 cases of hepatocellular carcinoma and 8 cases of focal nodular hyperplasia. Fibrolamellar carcinoma was characterized by the presence of large amounts of tenascin in tumor stroma and by the scarce expression of basement membrane components at the contact of neoplastic clusters. Like normal hepatocytes, neoplastic cells constantly expressed the alpha1 integrin chain, lacked the beta4 integrin chain, and coexpressed E-cadherin and the hepatocyte N-related cadherin. Abnormalities in the expression of cell adhesion molecules, including altered cadherin expression, alphaV integrin chain induction, and CD44 expression, were detected in the majority of cases. The composition of the tumor stroma and the pattern of expression of cell adhesion molecules in fibrolamellar carcinoma were reminiscent of those observed in grade III and grade IV hepatocellular carcinomas. Our results therefore show that, despite its slow local growth and good prognosis, fibrolamellar carcinoma expresses many characteristics usually associated with clinically aggressive malignancies. Further studies are needed to identify the factors responsible for the apparent dissociation between clinical behavior and biological characteristics in this tumor.