Biomedical subjects

C M Giachelli

Publications and source records attributed to C M Giachelli.

64 records · Page 4Linked to original sources

PubMed1994

Tubulointerstitial disease in glomerulonephritis. Potential role of osteopontin (uropontin).

Interstitial inflammation and tubular injury accompany most types of glomerulonephritis and are likely to mediate progressive renal injury. We hypothesized that the interstitial monocyte/macrophage accumulation in nephritis involves osteopontin, a cell attachment glycoprotein that avidly binds macrophages in vitro and induces a macrophage-rich infiltrate on subcutaneous injection in mice (Singh et al, J Exp Med, 1990, 171: 1931). In this study, we demonstrate that osteopontin messenger RNA and protein levels are up-regulated in a proportion of proximal and distal tubules in three experimental models of glomerulonephritis. In all three models, the expression of osteopontin initially precedes histological evidence of tubular injury, but is correlated with subsequent sites of monocyte/macrophage accumulation and tubular damage. Osteopontin expression also correlates with the severity of the tubulointerstitial injury, being greatest in amino-nucleoside nephrosis. These data suggest that 1) osteopontin is up-regulated in tubules in glomerular disease; 2) osteopontin may be important for macrophage accumulation at specific sites in diseased tissue; and 3) osteopontin may therefore have a role in the pathogenesis of the tubulointerstitial injury that accompanies glomerulonephritis.

Animals↗

PubMed1993

Osteopontin is elevated during neointima formation in rat arteries and is a novel component of human atherosclerotic plaques.

In an earlier report, we used differential cloning to identify genes that might be critical in controlling arterial neointima formation (Giachelli, C., N. Bae, D. Lombardi, M. Majesky, and S. Schwartz. 1991. Biochem. Biophys. Res. Commun. 177:867-873). In this study, we sequenced the complete cDNA and conclusively identified one of these genes, 2B7, as rat osteopontin. Using immunochemistry and in situ hybridization, we found that medial smooth muscle cells (SMC) in uninjured arteries contained very low levels of osteopontin protein and mRNA. Injury to either the adult rat aorta or carotid artery using a balloon catheter initiated a qualitatively similar time-dependent increase in both osteopontin protein and mRNA in arterial SMC. Expression was transient and highly localized to neointimal SMC during the proliferative and migratory phases of arterial injury, suggesting a possible role for osteopontin in these processes. In vitro, basic fibroblast growth factor (bFGF), transforming growth factor-beta (TGF-beta), and angiotensin II (AII), all proteins implicated in the rat arterial injury response, elevated osteopontin expression in confluent vascular SMC. Finally, we found that osteopontin was a novel component of the human atherosclerotic plaque found most strikingly associated with calcified deposits. These data implicate osteopontin as a potentially important mediator of arterial neointima formation as well as dystrophic calcification that often accompanies this process.

Amino Acid Sequence↗

PubMed1992

Rat carotid neointimal smooth muscle cells reexpress a developmentally regulated mRNA phenotype during repair of arterial injury.

Smooth muscle cells (SMCs) cultured from the neointima of injured rat carotid arteries have a different shape and organization in vitro than SMCs from the uninjured media. The morphology of neointimal SMCs from adult rats strongly resembles that of a subset of medial SMCs from 12-day-old rat pups. In the present study, we show that adult carotid neointimal SMCs in vitro express the platelet-derived growth factor (PDGF)-B gene but have little or no PDGF alpha-receptor mRNA. In contrast, medial SMCs from contralateral uninjured carotids, grown and passaged under identical conditions, contain abundant PDGF alpha-receptor mRNA but little or no PDGF-B mRNA. Transcript levels for PDGF-A or PDGF beta-receptor were not different in neointimal versus medial SMC cultures. The PDGF mRNA phenotype of adult neointimal SMCs strongly resembles that of an aortic medial SMC subset from newborn rat pups. Although intriguing, the differences in SMC phenotypes we observed in cell culture may depend on unique conditions in vitro and do not necessarily mean that analogous SMC diversity also exists in vivo. To address this question, we constructed and screened a SMC cDNA library for additional molecular markers of the common "pup-intimal" SMC phenotype. Two cDNA clones were identified whose cognate mRNA levels were developmentally regulated in rat aorta in vivo and were present at high levels in the adult carotid neointima formed 2 weeks after balloon catheter injury. Importantly, elevated levels of these two cognate mRNAs in carotid neointima compared with underlying media were maintained in cultures of neointimal versus medial SMCs in vitro. DNA sequence analysis indicated that the cDNA clones encoded rat tropoelastin and alpha 1 procollagen (type I). These results provide further evidence that neointima formation in the adult rat carotid artery depends on reexpression of an SMC phenotype or subpopulation with special properties characteristic of earlier stages of artery wall development. Our studies to date indicate that two of these special properties are paracrine growth factor production and extracellular matrix synthesis.

Age Factors↗

PubMed1991

Developmentally regulated cytochrome P-450IA1 expression in cultured rat vascular smooth muscle cells.

We have examined the expression of cytochrome P-450IA1 (CypIa1) in cultured rat arterial smooth muscle cells (SMC). We report here that newborn (pup) aortic and adult carotid neointimal SMC express 10-20-fold higher steady-state levels of CypIa1 mRNA compared with identically treated SMC derived from adult aorta or uninjured carotid media, respectively. Nuclear transcription experiments suggest that the difference in basal expression of CypIa1 reflects differences in transcription rates of the gene in these two cell types. Treatment of adult aortic SMC with cycloheximide dramatically elevated CypIa1 mRNA levels and had little effect on the amount of this mRNA in pup SMC. That the cycloheximide effect in adult SMC was due to enhanced transcription of the CypIa1 gene was demonstrated by 1) the ability of actinomycin D to completely block the cycloheximide-induced increase in steady-state mRNA levels, and 2) the increased transcription rate of the CypIa1 gene in cycloheximide-treated adult nuclei determined via nuclear transcription assays. Treatment of either cell type with beta-napthoflavone increased CypIa1 mRNA levels to a similar extent, suggesting that the differential response to cycloheximide was specific and not a general lack of pup SMC to respond to transcriptional inducers of the CypIa1 gene. Our data suggest that a labile repressor of the CypIa1 gene is present in cultured adult aortic SMC and is absent, inactive, or at a greatly reduced level in cultured pup SMC.

Animals↗

PubMed1989

Isolation and characterization of rat cytochrome P-450IIB gene family members. Use of the polymerase chain reaction to detect expression of a novel P-450 mRNA.

The rat cytochrome P-450IIB gene family consists of at least 10 members, 2 of which, P-450b and P-450e, have been well characterized and are activated transcriptionally by phenobarbital (PB) in the liver. The remaining genes in this family have not been studied extensively. In this report, data are presented that provide additional characterization of a P-450IIB gene, termed gene IV. The complete gene IV and flanking regions were isolated from a rat liver Charon 4A genomic library and subjected to a variety of analyses. Structural homology to the P-450b gene was confirmed by comparative restriction mapping and high stringency hybridization of gene IV fragments to probes comprising the entire cDNA for P-450b. The 5'-portion of gene IV, including its flanking region, was sequenced and contained an open reading frame for 58 amino acids which were 62% related to exon 1 of the P-450b/e genes. Typical TATA and CAAT promoter elements, as well as two Sp1 core sequences and a site related to a glucocorticoid responsive element were found in gene IV. Northern blot studies with an oligomer probe specific to gene IV sequence indicated a 4.3-kilobase pair polyadenylated transcript present at low levels in untreated rat liver and inducible approximately 6-fold by PB treatment. Primer extension experiments indicated that the transcription initiation site mapped to a position on gene IV that was analogous to that reported for the structurally similar P-450e gene. Due to the low levels of hepatic RNA expression, we employed the polymerase chain reaction to facilitate characterization of gene IV transcripts. The polymerase chain reaction data verified that gene IV transcripts were elevated after PB treatment. Significantly, the polymerase chain reaction results demonstrated that gene IV transcripts were associated with hepatic polysome fractions, indicating their active utilization in this tissue. Polymerase chain reaction analysis of RNA also indicated constitutive expression of gene IV transcripts in rat lung, kidney, and testis, as well as fetal rat liver. Together, these data show that gene IV is a transcribed member of the P-450IIB gene family and, like the well characterized P-450b and P-450e genes, is positively regulated by PB in rat liver.

Animals↗

PubMed1987

Developmental regulation of cytochrome P-450 genes in the rat.

Synthetic oligomer probes were used in hybridization experiments to investigate the developmental regulation of cytochrome P-450 (P-450) genes in rat liver. Transplacental induction by phenobarbital of P-450b and P-450e mRNAs was not detectable in fetal rat livers prior to day 21 of gestation. The levels of these mRNAs increased approximately 2-fold from gestational day 21 to day 22 in phenobarbital-induced liver. P-450b and P-450e mRNAs were shown to be adenylated and the fractions associated with polysomes were similar in both fetal and adult livers. No P-450b or P-450e mRNAs were detected in fetal lung and kidney RNA preparations regardless of pretreatment. Southern blot data utilizing fetal liver DNA suggests that responsiveness to xenobiotic induction during development is not attained by rearrangement of P-450b or P-450e genes. Experiments with probes specific for P-450c and P-450d failed to detect their respective mRNAs in fetal livers from 3-methylcholanthrene (3-MC)-treated or untreated rats. Both species were detectable in 3-MC-treated rats 1 week after birth. The levels of 3-MC-inducible P-450c and P-450d mRNAs increased with age and peaked approximately 3 weeks after birth. Hepatic P-450d mRNA levels in 3-MC-treated or control rats was consistently higher than P-450c mRNA levels at all ages studied.

Animals↗

PubMed1986

Regulation of cytochrome P-450b and P-450e mRNA expression in the developing rat. Hybridization to synthetic oligodeoxyribonucleotide probes.

We conducted solution hybridization and Northern blot experiments utilizing synthetic 18'-mer oligodeoxyribonucleotides complementary to two major rat hepatic phenobarbital-inducible cytochrome P-450s, P-450b and P-450e, to assess their mRNA expression during rat development. At all ages studied, with one exception (i.e. in day 22 neonates), P-450b mRNA was not detected in control animals. However, traces of P-450e message were observed in control animals on day 22 and persisted to adulthood. Phenobarbital pretreatment caused marked increases in hepatic mRNA for both P-450s as early as 22 days after conception. No increases were observed in RNA isolated from phenobarbital-pretreated day 10 or 19 rats. In general, the inducible levels of P-450b and P-450e mRNA increased as a function of age. The age-dependent increase in responsiveness to phenobarbital was associated with an age-dependent decrease in the ratio of P-450b to P-450e mRNA levels. The levels of P-450b/P-450e varied from a ratio of 19 at day 22 of development to a ratio of 5 at day 62 of development. Maximal levels of phenobarbital-induced hepatic RNA for both isozymes occurred 24 days after birth (day 46 of development), at which time P-450b and P-450e mRNAs accumulated to levels 2.4- and 1.8-fold greater, respectively, than levels found in comparably induced adult rat liver. Northern blot analyses indicated that the major mRNA species hybridizing to either the P-450b or P-450e oligomers in all age groups studied was approximately 1.8 kilobases.

Animals↗

PubMed1986

Carbon monoxide inhibits monooxygenation by the conceptus and embryotoxic effects of proteratogens in vitro.

Evidence that Day 10 to 11 rat conceptuses can contain sufficient quantities of xenobiotic-oxidizing P-450(s) to catalyze the conversion of proteratogens to reactive teratogenic intermediates is presented. Quantities of reactive intermediates generated by conceptuses from 2-acetylaminofluorene (AAF) were sufficient to result in the appearance of gross morphologic abnormalities with a high incidence of neural tube anomalies. Carbon monoxide effectively inhibited the conversion of AAF to hydroxylated metabolites by rat conceptuses, and also inhibited the capacity of AAF to elicit neural tube anomalies in cultured embryos. Inhibition by CO of either parameter was observed only with conceptuses exposed in utero to the potent P-450 inducer, 3-methylcholanthrene.

2-Acetylaminofluorene↗

PubMed1985

Effects of 3-methylcholanthrene and phenobarbital on the capacity of embryos to bioactivate teratogens during organogenesis.

Pregnant Sprague-Dawley rats were divided into four groups and given ip injections of 3-methylcholanthrene (MC) in corn oil, corn oil only, phenobarbital (PB) in Hank's balanced salt solution (HBSS), or HBSS only. Maternal animals were killed on Day 10 of gestation, and embryos from each group were explanted in medium containing cyclophosphamide (CP), 2-acetylaminofluorene (AAF), or dimethylsulfoxide vehicle. After a 24-hr culture period, embryos from dams treated with HBSS, corn oil, or PB/HBSS exhibited no increase in abnormalities (as compared with controls) when either CP or AAF were added to the media. However, embryos transplacentally preexposed to MC and subsequently treated during culturing with AAF (but not CP) exhibited striking increases in malformation incidence. Commonly observed malformations included abnormally open neural tubes, abnormal flexure rotation, and prosencephalic defects. Homogenates of Day 10 embryos transplacentally preexposed to MC exhibited readily measurable oxidative biotransformation of AAF as assessed with HPLC. Biotransformation of AAF by embryos from the other three groups was virtually undetectable. Incorporation of exogenously supplemented bioactivating systems from livers of mature animals indicated that postmitochondrial supernatant fractions (S-9) from male, MC-pretreated rats effectively catalyzed the conversion of AAF (but not CP) to embryotoxic metabolites. Conversely, hepatic S-9 from adult, male, PB-pretreated rats was highly effective in converting CP (but not AAF) to embryotoxic metabolites. The results indicated the inducerspecific occurrence of embryonic bioconversion of AAF to embryotoxic metabolites via MC-inducible, P-450-dependent, embryonic enzyme systems.

2-Acetylaminofluorene↗

PubMedDate not supplied

A functional role for osteopontin in experimental crescentic glomerulonephritis in the rat.

This study examined whether osteopontin (OPN), a molecule with monocyte chemotactic and adhesive activity, participates in macrophage-mediated renal disease, Accelerated anti-glomerular basement membrane glomerulonephritis was induced in groups of six rats. Animals were treated with a neutralizing anti-OPN or an irrelevant control antibody over days 0-7 (induction phase) or days 7-14 (established disease). Administration of the control antibody had no effect on the severity of the disease. In contrast, anti-OPN treatment significantly reduced glomerular injury (urinary protein excretion) and prevented a loss of renal function (creatinine clearance) during the induction of disease. This was accompanied by a significant reduction in renal macrophage and T-cell accumulation, T-cell activation, and histological injury (glomerular hypercellularity, segmental lesions, crescents, and tubulointerstitial lesions). An important finding was that anti-OPN treatment of established crescentic glomerulonephritis led to a significant reduction in glomerular injury and recovery of renal function in association with inhibition of macrophage and T-cell accumulation, T-cell activation, and histological damage. Anti-OPN treatment significantly inhibited the upregulation of OPN and its ligand CD44 but demonstrated no effect on upregulation of intercellular adhesion molecule-1 (ICAM-1) expression in the kidney. Interestingly, anti-OPN treatment significantly reduced skin swelling and leukocyte infiltration in the delayed type hypersensitivity response. However, anti-OPN treatment had no effect on the humoral immune response. In summary, this study has demonstrated that OPN plays a functional role in macrophage and T-cell accumulation and renal damage in both the induction and progression of a rat model of crescentic glomerulonephritis. Thus, OPN may be of pathological importance in human glomerulonephritis and in cell-mediated immune diseases generally.

Animals↗