Lipids are an important source of chemical modification of tissue proteins, even in the absence of hyperglycemia. PM inhibited AGE/ALE formation and hyperlipidemia and protected against renal and vascular pathology in a nondiabetic model.
The cardiac extracellular matrix, composed predominantly of collagenous fibers, forms a stress-tolerant network that facilitates the distribution of forces generated in the heart and provides for proper alignment of cardiac myocytes. Although considerable information exists regarding the morphological organization of the heart extracellular matrix, little is known about the regulation of the synthesis and accumulation of extracellular matrix components. A potentially significant factor in the cardiovascular system is mechanical stimulation including changes in physical tension and pressure. We recently have developed an in vitro model system to elucidate the effects of mechanical stretch on isolated populations of heart cells. In the present study, we have used biochemical and molecular biological techniques to analyze changes in collagen synthesis by cardiac fibroblasts in response to mechanical stretch. These studies show that the ratio of collagen type III to collagen type I increases in mechanically stretched cells. They also show that type III collagen mRNA levels are increased in response to cyclic mechanical stretch for durations as short as 12 hours. Type I collagen mRNA levels were not found to change under the stretch conditions used in this study. Our results emphasize the potential regulatory role of mechanical stimulation in the expression of specific genes in the heart and support previous studies indicating this to be an intriguing in vitro model of cardiac hypertrophy.
This study has examined the role of galectin-3 (GaL3), a multicompartmented N-acetyllactosamine-binding chimeric lectin, on atherogenesis in the ApoE-deficient mouse model of atherosclerosis. Pathological changes consisting of atheromatous plaques, atherosclerotic microaneurysms extending into periaortic vascular channels, and adventitial and periaortic inflammatory infiltrates were assessed in an equal number (n ؍ 36) of apolipoprotein (Apo)E-deficient mice and ApoE-GaL3 double-knockout mice. These mice were divided into three age groups, 21 to 23 weeks, 25 to 31 weeks, and 36 to 44 weeks of age. Results of this morphological analysis have shown an age-related increase in the incidence of aorta atheromatous plaques and periaortic vascular channels in ApoEdeficient mice. By contrast ApoE/GaL3 double-knockout mice did not show an increase in pathological changes with age. The 36-to 44-week group of ApoE
To study the signaling pathway involved in the regulation of galectin-3 expression we used phorbol ester to stimulate macrophage differentiation of THP-1 cells. Treatment with phorbol 12-myristate 13-acetate (PMA) increased significantly the level of expression of galectin-3 in THP-1 cells. PMA-induced galectin-3 overexpression was blocked by: protein kinase C inhibitors staurosporine, calphostin C, and apigenin; tyrosine-specific protein kinase inhibitors genistein and tyrphostin A25; PD 98059, a selective inhibitor of mitogen-activated protein kinase (MAPK) kinase 1 (MEK1 or MKK1); and SB 203580, a specific inhibitor of p38 MAPK. Galectin-3 up-regulation was not affected by exposure to two inhibitors of cAMP-dependent protein kinase (PKA), H-89 and KT5720. Co-transfection of pPG3.5, a plasmid vector containing the rabbit galectin-3 promoter and the constructs pMCL-MKK1 N3 or pRC-RSV-MKK3Glu that constitutively express MKK1 and MKK3, raised the activity of galectin-3 promoter by 185% and 110%, respectively. Co-transfection with a Ha-Ras expression vector stimulated galectin-3 promoter activity approximately 10-fold. Expression of c-Jun or v-Jun raised the level of galectin-3 promoter activity more the three- and fourfold, respectively. Co-transfection of c-Jun and pPG3.5 5'-upstream deletion mutants resulted in a reduction of the galectin-3 promoter activity by 50% to 80%. Transfection of c-Jun, v-Jun or Ha-Ras increased significantly galectin-3 protein in THP-1 cells. These findings indicated that Ras/MEKK1/MKK1-dependent/AP-1 signal transduction pathway plays an important role in the expression of galectin-3 in PMA-stimulated macrophages. We further investigated the effect of modified lipoproteins on galectin-3 expression in macrophages. Murine resident peritoneal macrophages loaded with acetylated low-density lipoprotein (AcLDL) or oxidized LDL (OxLDL) showed increased galectin-3 protein and mRNA. These results showed that treatment of macrophages with PMA or modified lipoproteins results in galectin-3 overexpression. These findings may explain the enhanced expression of galectin-3 in atherosclerotic foam cells and suggest that Ras/MAPK signal transduction pathway is involved in controlling this gene.
A spontaneously arising continuous cell line (Rb-1) derived from collagenase-elastase digested rabbit aorta has been propagated in vitro for over 100 passages. During this period, the Rb-1 cells remained spindle-shaped and formed regularly oriented parallel bundles. After Passage 50, Rb-1 cells were found to be serum-independent in their growth and reached higher saturation density than rabbit aorta smooth muscle cells. Alpha-actin and desmin filaments were detected by immunostaining in Rb-1 cells and early passage of rabbit aorta smooth muscle cells. The proportion of alpha-actin transcripts in Rb-1 cells was lower than that of transcripts for beta- and gamma-actins. The modal chromosome number was maintained at 44 between Passages 11 and 60, and two marker chromosomes were constantly present. Infection of Rb-1 cells with two strains of herpes simplex virus type 1 resulted in high titers of virus, whereas a herpes simplex virus type 2 temperature-sensitive mutant replicated only at the permissive temperature. The Rb-1 cell line could be used for the study of vascular smooth muscle cell proliferation and their interaction with viruses.
Three rat ventral prostate (RVP) cell lines transformed after in vitro treatment with cadmium chloride (CdCl2) and one control untreated cell line were tested for tumorigenicity in newborn rats. All three cadmium-transformed RVP cell lines induced tumors at the site of inoculation in 95-100% of animals. The fibroblastoid RVP56Cd cell line induced sarcomas, whereas the epithelial cell lines RVP47-3G and RVP47-3F produced highly differentiated squamous cell carcinomas. About 20% of animals injected with RVP47-3G developed lung and splenic metastases. The tumors could be further passaged into young rats. The sarcomas had a hyperdiploid modal chromosome number similar to that of the RVP56Cd cell line. Carcinomas induced by the RVP47-3G cell line had a large proportion of stromal metaphases. The modal chromosome number of these carcinomas was in the hypertriploid-hypotetraploid range, similar to that of the parental cell line. These results demonstrate that treatment of RVP cells with CdCl2 in vitro results in neoplastic transformation. Since both fibroblastoid and epithelial prostate cells have undergone transformation, it seems possible that cadmium acted as a carcinogen without cell specificity. The susceptibility of these cells to the carcinogenic effect may be related to their resistance to cadmium. In the process of neoplastic transformation induced by CdCl2 in RVP epithelial cells changes of squamous metaplasia occur, and probably precede acquisition of tumorigenicity.
Large, negatively charged, multilamellar liposomes were examined for their ability to improve the therapeutic activity of the broad-spectrum antiviral agent ribavirin (RIB) and the synthetic immunostimulant muramyl tripeptide (MTP-PE) in the treatment of viral pneumonitis. Liposome-encapsulated MTP-PE (L-MTP-PE) was superior to free MTP-PE in activating alveolar macrophages and in protecting mice against intranasal challenge with 10 LD50 (50% lethal dose) of herpes simplex virus type 1 (HSV-1). Mice treated with liposome-encapsulated or free MTP-PE had no detectable viremia and had lower pulmonary titers of virus than controls. Liposome-encapsulated RIB (L-RIB; 3 mg per mouse), administered several hours after infection, was more effective than was free RIB (10 mg per mouse) in protecting mice against intranasal challenge with 10 LD50 of influenza virus, but neither L-RIB nor free RIB protected mice against HSV-1 infection. In contrast, combination therapy with both L-RIB and L-MTP-PE was more effective than either agent used alone.
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