Maurice Nachtigal

Collagen expression in mechanically stimulated cardiac fibroblasts.

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.

Oncogenicity of rat prostate cells transformed in vitro with cadmium chloride

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.

Characterization of a continuous smooth muscle cell line derived from rabbit aorta

SummaryA 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 the 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 simples virus type 1 resulted in high titers of virus, whereas a herpes simplex virus type 2 temperature-sensitive mutant replication 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.

Amplification of stromelysin-3 transcripts from carcinomas of the colon☆

Stromelysin-3 has been recently described in association with the stroma of different types of cancer including colorectal carcinomas. This article reports the detection of transcripts for stromelysin-3 (matrix metalloproteinase-11 [MMP-11]) in extracts of tissue from colorectal carcinomas using the technique of reverse transcription-polymerase chain reaction (RT-PCR). In 12 cases of primary colon carcinoma, stromelysin-3 messenger RNA (mRNA) was detected after 25 cycles, whereas this procedure did not reveal stromelysin-3 mRNA expression in one rectal carcinoma micrometastasis to the liver or in normal colon tissue (controls) after 30 cycles of PCR. However, stromelysin-3 mRNA was detected in normal colon specimens after 45 cycles. The high sensitivity of this technique allows application for the investigation of the expression of stromelysin-3 in small amounts of tissue.

Transformation of prostatic epithelial cells and fibroblasts with cadmium chloride in vitro

Primary cultures of fibroblasts and epithelial cells were established from rat ventral prostate (RVP), canine (CP), baboon (BP), and human (HP) prostates, and were used in an assay system to evaluate cadmium chloride (CdCl2) cytotoxicity in vitro. Fibroblasts were always more susceptible to CdCl2 cytotoxicity than the epithelial cells of the same species. There was a distinct species variability to CdCl2 cytotoxicity, with RVP cells being greater than 200 times more susceptible than HP. Primary cultures treated with CdCl2 were subcultivated to establish cell lines. Only RVP fibroblast and epithelial cells resulted in permanent cell lines. Two fibroblast and two epithelial cell lines were derived from CdCl2-treated RVP cell cultures. The epithelial cell lines possessed tonofilaments, desmosomes and keratin. All four cell lines were resistant to CdCl2, had different karyotypes and an excess of chromosome 13. These results demonstrate the transforming potential of cadmium on prostate cells. The role of metallothionein and the significance of extra chromosomes 13 are discussed as possible factors of cadmium resistance.

Glycofection: The Ubiquitous Roles of Sugar Bound on Glycoplexes

Glycofection (transfection by using sugar-substituted polylysine) was assessed in order to provide an alternative to viral vectors for the transfer of genes into vascular smooth muscle cells. A rabbit vascular smooth muscle cell line (Rb-1 cells) was selectively transfected by using glycoplexes (glycosylated polylysine/pSV2LUC complexes) in the presence of 10 mu M of the fusogenic peptide GALA. A sugar-specific transfection was obtained when the glycofection was conducted for 1 h with glycoplexes containing either alpha Gal, alpha -Glc, alpha -GalNAc, beta -GlcNAc, or beta -GalNAc residues. The gene expression was high after transfection, with glycoplexes bearing alpha Gal, alpha -GalNAc, or beta -GalNAc residues that were weakly internalized, and low with glycoplexes carrying Lact or Rha residues that were well taken up by cells. These results suggest that 1) glycofection can be a good approach for a selective transfer of genes intovascular smooth muscle cells, 2) an efficient uptake of the glycoplexes i...

Ventricular collagen matrix and alterations.

There is a complex extracellular structural matrix in the heart. This matrix appears to be composed of a variety of fibrils and fibers extending from the cell surface to the basal lamina and from the basal lamina to the matrix. The extensions into the extracellular region interconnect with a system of collagen bundles. The latter are so located that they would tether the myocytes to each other as well as tether the capillaries to the myocetes. There is an extensive weave of collagen analogous to the perimysium of skeletal muscle that separates groups of myocytes. The weave surrounding a group of myocytes is connected to adjacent weave patterns by long, tendonlike structures. The collagen matrix around cells disappears 2-3 hr after coronary-artery occlusion. In the periinfarct region of viable cells, the matrix is similarly lost and is replaced by scarlike collagen. Encephalomyocarditis virus causes a similar loss of the matrix in necrotic as well as some adjacent nonnecrotic regions. Replacement of the lost matrix is by scar tissue. The long-term appearance of the replacement fibrosis closely resembles the appearance of diffuse fibrosis as seen in a variety of conditions. These observations suggest that diffuse fibrosis can occur secondary to loss of the matrix both with and without myocyte necrosis. This may help explain the diffuse left ventricular fibrosis as seen in a variety of human disease.

Immortalization of Rabbit Vascular Smooth Muscle Cells after Transfection with a Fragment of the BglII N Region of Herpes Simplex Virus Type 2 DNA

Rabbit arterial smooth muscle cells were transfected with the pBC24neo plasmid DNA which consists of a sequence of 790 base pairs from the BglII N fragment of herpes simplex virus type 2 DNA linked to the neo resistance gene. Selection in G418-containing medium resulted in eleven immortalized clones which showed increased growth rate and saturation densities. One of the eleven clones maintained the transforming DNA sequence integrated in the cellular genome and showed continuous resistance to G418, but Southern blot analysis of the other ten immortalized clones did not detect pBC24neo DNA sequences. Possible mechanisms of herpes simplex virus induced immortalization and their implications in the development of atheromatous plaques are discussed.

Chromosome changes in rat embryo cell lines transformed by temperature-sensitive mutants and sheared DNA of herpes simplex virus

The chromosomes of six rat embryo cell lines transformed with herpes simplex virus (HSV) temperature-sensitive (ts) mutants were examined at different passages of in vitro cultivation. Two cell lines were predominantly diploid, one cell line was hyperdiploid, one cell line was pseudodiploid, and two cell lines were hypotetraploid. In near-diploid cell lines chromosome No. 9 was most frequently involved in chromosome changes. All three cell lines derived from tumors obtained after one transplantation of HSV-transformed cells into baby rats were pseudodiploid, but each had different marker chromosomes. Chromosome No. 15 was involved in the formation of two out of four marker chromosomes. Four cell lines derived from tumors developing after two and three transplantations were hypodiploid and showed large chromosome variation. The occurrence of 25 marker chromosomes in three tumor-derived cell lines resulted in gains in parts from chromosomes No. 2, 6, and 7. One marker chromosome had a homogeneously faintly stained region. Chromosomes No. 2, 3, 7, and 12 were more frequently involved in the formation of marker chromosomes. No chromosome change was found to be specifically associated with HSV-induced transformation of rat cells, but chromosome changes in tumor-derived cell lines may provide selective advantage for survival and autonomous growth in the host animal.

Transformation of rabbit arterial smooth muscle cells with simian virus 40.

SummaryIn vitro studies were carried out to induce viral transformation of vascular smooth muscle cells. Cultured rabbit arterial smooth muscle cells were infected with simian virus 40 (SV 40), and transformed cultures were produced that exhibit altered morphology, increased growth rate and plating efficiency, growth on semi-solid substrate, and chromosomal abnormalities. Nuclear SV 40 T-antigen was detected in all cells of these cultures. Muscle-specific actin was identified by a specific monoclonal antibody suggesting retention of smooth muscle cell characteristics by the transformed cells. Significant cytoplasmic lipid accumulation occurred in transformed cells incubated with β-very low density lipoprotein, as revealed both by chemical analyses and Nile Red lipid staining of the cultures. The transformed smooth muscle cells grow permanently in cell culture. Our investigations show that arterial smooth muscle cells transformed with SV 40 virus exhibit altered phenotypic properties distinct from that of normal arterial smooth muscle cells.