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Biomedical subjects

O Kocher

Publications and source records attributed to O Kocher.

At least 37 records · Page 2Linked to original sources
PubMed1990

Transforming growth factor beta 1 modulates extracellular matrix organization and cell-cell junctional complex formation during in vitro angiogenesis.

Transforming growth factor-beta 1 (TGF-beta 1) is angiogenic in vivo. In two-dimensional (2-D) culture systems microvascular endothelial cell proliferation is inhibited up to 80% by TGF-beta 1; however, in three-dimensional (3-D) collagen gels TGF-beta 1 is found to have no effect on proliferation while eliciting the formation of calcium and magnesium dependent tube-like structures mimicking angiogenesis. DNA analyses performed on 3-D cell cultures reveal no significant difference in the amount of DNA or cell number in control versus TGF-beta 1 treated cultures. In 2-D cultures TGF-beta 1 is known to increase cellular fibronectin accumulation; however, in 3-D cultures no difference is seen between control and TGF-beta 1 treated cells as established by ELISA testing for type IV collagen, fibronectin, and laminin. In 3-D cultures there is increased synthesis and secretion of type V collagen in both control and TGF-beta 1 treated cultures over 2-D cultures. Even though an equal amount of type V collagen is seen in both 3-D conditions, there is a reorganization of the protein with concentration along an organizing basal lamina in TGF-beta 1 treated cultures. EM morphological analyses on 3-D cultures illustrate quiescent, control cells lacking cell contacts. In contrast, TGF-beta 1 treated cells show increased pseudopod formation, cell-cell contact, and organized basal lamina-like material closely apposed to the "abluminal" plasma membranes. TGF-beta 1 treated cells also appear to form junctional complexes between adjoining cells. Immunofluorescence using specific antibodies to the tight junction protein ZO-1 results in staining at apparent cell-cell junctions in the 3-D cultures. Northern blots of freshly isolated microvascular endothelium, 2-D and 3-D cultures, using cDNA and cRNA probes specific for the ZO-1 tight junction protein, reveal the presence of the 7.8 kb mRNA. Western blots of rat epididymal fat pad endothelial cells (RFC) monolayer lysates probed with anti-ZO-1 label a 220 kd band which co-migrates with the bonafide ZO-1 protein. These data confirm and support the hypothesis that TGF-beta 1 is angiogenic in vitro, eliciting microvascular endothelial cells to form tube-like structures with apparent tight junctions and abluminal basal lamina deposition in three-dimensional cultures.

Animals↗
PubMed1990

Alternative splicing of endothelial cell fibronectin mRNA in the IIICS region. Functional significance.

Transforming growth factor-beta 1 (TGF-beta 1) is thought to play a role in modulating vascular cell function in vivo. In vitro, it decreases endothelial cell proliferation and migration. We postulated that these biologic activities could be mediated through TGF-beta 1 modulation of specific gene expression. Therefore we differentially screened a human umbilical vein endothelial cell cDNA library with cDNAs prepared from both untreated and TGF-beta 1-treated bovine aortic endothelial cells. Using this technique, we isolated many TGF-beta 1-induced cDNA clones. Sequence analysis of these cDNAs showed that many of them corresponded to alternatively spliced fibronectin mRNAs. These fibronectin clones all contained the extradomain I (ED I) but three different forms of the type III connecting segment (IIICS). These different fibronectin cDNAs were expressed in bacteria and the recombinant proteins used to study the effects of IIICS alternative splicing on cell attachment, spreading, and migration in bovine aortic endothelial and smooth muscle cells and B16F10 melanoma cells. The results of these experiments show that attachment and spreading of bovine aortic endothelial and smooth muscle cells depend primarily on the presence of the Arg-Gly-Asp-Ser (RGDS) sequence in the recombinant fibronectin proteins. However attachment and spreading of bovine aortic endothelial cells are modulated by alternative splicing in the IIICS region. Specifically splicing of the IIICS region decreases spreading and increases migration rates of the endothelial cells. On the contrary, using a cell line (B16F10 melanoma cells) that is known not to require the RGDS sequence for adhesion confirmed previous findings that B16F10 melanoma cells do not require the presence of the RGDS sequence for attachment and spreading. Indeed B16F10 cells were able to attach and spread on two recombinant proteins that did not contain the RGDS sequence. However attachment and spreading of B16F10 were dramatically inhibited when a 75-base pair DNA fragment was removed from the 5' end of the IIICS region. These results suggest that various regions of the fibronectin molecule may be able to interact with different cell populations to promote cell attachment and spreading, and that alternative splicing may modulate this process.

Animals↗
PubMed1989

Modulation of actin mRNAs in cultured vascular cells by matrix components and TGF-beta 1.

Alpha-smooth muscle actin is currently considered a marker of smooth muscle cell differentiation. However, during various physiologic and pathologic conditions, it can be expressed, sometimes only transiently, in a variety of other cell types, such as cardiac and skeletal muscle cells, as well as in nonmuscle cells. In this report, the expression of actin mRNAs in cultured rat capillary endothelial cells (RFCs) and aortic smooth muscle cells (SMCs) has been studied by Northern hybridization in two-dimensional cultures seeded on individual extracellular matrix proteins and in three-dimensional type I collagen gels. In two-dimensional cultures, in addition to cytoplasmic actin mRNAs which are normally found in endothelial cell populations, RFCs expressed alpha-smooth muscle (SM) actin mRNA at low levels. alpha-SM actin mRNA expression is dramatically enhanced by TGF-beta 1. In addition, double immunofluorescence staining with anti-vWF and anti-alpha-SM-1 (a monoclonal antibody to alpha-SM actin) shows that RFCs co-express the two proteins. In three dimensional cultures, RFCs still expressed vWF, but lost staining for alpha-SM actin, whereas alpha-SM actin mRNA became barely detectable. In contrast to two-dimensional cultures, the addition of TGF-beta 1 to the culture media did not enhance alpha-SM actin mRNA in three-dimensional cultures, whereas it induced rapid capillary tube formation. Actin mRNA expression was modulated in SMCs by extracellular matrix components and TGF-beta 1 with a pattern very different from that of RFCs. Namely, the comparison of RFCs with other cell types such as bovine aortic endothelial cells shows that co-expression of endothelial and smooth muscle cell markers is very unique to RFCs and occurs only in particular culture conditions. This could be related to the capacity of these microvascular endothelial cells to modulate their phenotype in physiologic and pathologic conditions, particularly during angiogenesis, and could reflect different embryologic origins for endothelial cell populations.

Actins↗
PubMed1989

The interactions of vascular cells with solid phase (matrix) and soluble factors.

The vessel wall is composed of heterogeneous cell populations residing in a variety of vascular beds. Each cell type has different functions and morphologies but all of them have a role in the repair process following vascular injury. Responses to injury vary depending upon the type and extent of the injury and the vascular bed affected. The sheet migration and proliferation exhibited by large vessel endothelial cells is in striking contrast to the migration through soft tissues and tube formation exhibited by microvascular endothelial cells in response to injury. Vascular smooth muscle cells respond to injury by migrating into the intima, proliferating and synthesizing matrix, causing intimal thickening. The response to injury by vascular cells appears to be modulated, in part, by the composition and organization of the surrounding matrix and the various platelet factors and cytokines found at sites of injury. Furthermore, evidence has been accrued in culture, suggesting that solid phase (matrix) and soluble factors modulate each other's effects on local vascular cell populations following injury.

Animals↗
PubMed1989

Endothelial cell behavior after denudation injury is modulated by transforming growth factor-beta1 and fibronectin.

Endothelial denudation injury after endarterectomy, autologous and synthetic grafting and balloon angioplasty leads to exposure of thrombogenic vessel wall material and may elicit an atherogenic response in the media of the affected vessels in which complete reendothelialization may not occur. While the role(s) of extracellular matrix composition and organization in this process are only incompletely understood, it is widely accepted that endothelial cells respond to matrix components in specific, complex fashions. In this report we demonstrate that large vessel endothelial cell migration is affected by the surrounding matrix and the soluble factor, transforming growth factor-beta1, which may mediate its effects, in part, by modulating endothelial cell matrix synthesis. Specifically, large vessel endothelial cell migration is decreased on a fibronectin substratum and in the presence of transforming growth factor-beta1, which increases fibronectin mRNA and protein accumulation in culture. Inhibition of sheet migration is also elicited by the addition of soluble fibronectin to the cultures. These in vitro findings are consistent with our in vivo findings of increased staining of fibronectin luminally and in the intima in the chronically deendothelialized region of a balloon catheter denuded carotid artery. Thus, reendothelialization after iatrogenic and natural injury appears to be a complex process which can be modulated by the underlying matrix and soluble factors, which may themselves modulate the matrix synthesis of local vascular cells.

Animals↗
PubMed1988

Localization of T lymphocytes and macrophages in fibrous and complicated human atherosclerotic plaques.

The cellular composition of aortic atherosclerotic plaques was analyzed by immunocytochemistry using cell type-specific monoclonal antibodies. T lymphocytes and monocytes/macrophages were detected both in early, fibrous plaques, and in more advanced, complicated ones. Many smooth muscle cells in these plaques expressed the class II MHC antigen, HLA-DR. Since this antigen is inducible by T cell products, our findings suggest that T cell-smooth muscle interactions occur during atherogenesis.

Antibodies, Monoclonal↗
PubMed1988

Arterial smooth muscle cells in vivo: relationship between actin isoform expression and mitogenesis and their modulation by heparin.

Quiescent smooth muscle cells (SMC) in normal artery express a pattern of actin isoforms with alpha-smooth muscle (alpha SM) predominance that switches to beta predominance when the cells are proliferating. We have examined the relationship between the change in actin isoforms and entry of SMC into the growth cycle in an in vivo model of SMC proliferation (balloon injured rat carotid artery). alpha SM actin mRNA declined and cytoplasmic (beta + gamma) actin mRNAs increased in early G0/G1 (between 1 and 8 h after injury). In vivo synthesis and in vitro translation experiments demonstrated that functional alpha SM mRNA is decreased 24 h after injury and is proportional to the amount of mRNA present. At 36 h after injury, SMC prepared by enzymatic digestion were sorted into G0/G1 and S/G2 populations; only the SMC committed to proliferate (S/G2 fraction) showed a relative slight decrease in alpha SM actin and, more importantly, a large decrease in alpha SM actin mRNA. A switch from alpha SM predominance to beta predominance was present in the whole SMC population 5 d after injury. To determine if the change in actin isoforms was associated with proliferation, we inhibited SMC proliferation by approximately 80% with heparin, which has previously been shown to block SMC in late G0/G1 and to reduce the growth fraction. The switch in actin mRNAs and synthesis at 24 h was not prevented; however, alpha SM mRNA and protein were reinduced at 5 d in the heparin-treated animals compared to saline-treated controls. These results suggest that in vivo the synthesis of actin isoforms in arterial SMC depends on the mRNA levels and changes after injury in early G0/G1 whether or not the cells subsequently proliferate. The early changes in actin isoforms are not prevented by heparin, but they are eventually reversed if the SMC are kept in the resting state by the heparin treatment.

Actins↗
PubMed1987

Analysis of alpha-smooth-muscle actin mRNA expression in rat aortic smooth-muscle cells using a specific cDNA probe.

We constructed two cDNA probes, the first of which hybridizes with all rat actin mRNAs while the second is specific for alpha-smooth muscle (SM) actin mRNA. Northern hybridization using these probes showed that, in normal rat aortic media, the proportion of alpha-SM actin mRNA expression increases during development, reaching about 90% of the total actin mRNA level in adult animals. As compared to the situation in normal aortic media, the proportion of alpha-SM actin mRNA was found to decrease significantly in intimal thickening 15 days after endothelial injury, i.e. when SM cells (SMCs) are actively replicating. At 60 days after injury, the SMCs were observed to have stopped dividing and to have recovered a normal content of alpha-SM actin mRNA. The content of alpha-SM actin mRNA was also selectively decreased (as compared to controls) in the hypotensive abdominal aortic media located below an aortic ligature, while it was not modified in the thoracic hypertensive segment above the same ligature. Primary cultures of rat aortic SMCs synthesize and contain low amounts of alpha-SM actin, but their alpha-SM actin mRNA content is similar to that of SMCs in vivo. As compared to primary cultures, the proportion of alpha-SM actin mRNA was found to be significantly decreased in SMCs at the fifth passage, at which stage it became comparable to the level of synthesized alpha-SM actin. Thus, the synthesis and expression of alpha-SM actin in SMCs appear to be regulated predominantly at the level of gene transcription in certain situations (e.g. aortic ligature in vivo and culture at the fifth passage), and predominantly at a post-transcriptional level in other situations (e.g. primary culture).

Actins↗
PubMed1986

Cytoskeletal features of normal and atheromatous human arterial smooth muscle cells.

The distribution of actin, vimentin, desmin, and tropomyosin was studied in the media of the human aorta and femoral and coronary arteries, as well as in atheromatous plaques from the same arteries, by means of immunofluorescence, densitometric analysis of sodium dodecylsulfate-polyacrylamide gel electrophoresis, and bidimensional gel electrophoresis. The proportions of desmin-containing cells varied in the media of different arteries; 4 per cent of the cells in the aorta, 11 per cent in the coronary artery, and 37 per cent in the femoral artery contained desmin. In fibrous atheromatous plaques, independently of the artery, desmin-containing cells were almost absent, but they reappeared in complicated lesions. The content of vimentin per smooth muscle cell increased in fibrous atheromatous plaques, whereas the content of actin and tropomyosin was less than in normal media. Moreover, the alpha-actin predominance observed in the media was transformed to beta-actin predominance in the atheromatous plaques. These cytoskeletal changes provide new, possibly useful, biochemical markers for the characterization of smooth muscle cells during early and advanced phases of atheroma formation.

Adult↗
PubMed1986

Expression of actin mRNAs in rat aortic smooth muscle cells during development, experimental intimal thickening, and culture.

The expression of actin-isoform mRNAs in the smooth muscle cells (SMC) of the aortic media in rats has been studied by Northern-blot hybridization, using a general actin-cRNA probe, and two cRNA probes specific for beta- and gamma-cytoplasmic actins, during: (1) development, (2) intimal thickening after endothelial injury induced by balloon catheterization, and (3) growth in culture. In 5-day-old rats, the ratio between alpha-smooth-muscle-actin mRNA and beta- and gamma-cytoplasmic-actin mRNAs was close to 1. It increased to about 4 in 6-week-old rats. Replicating SMC from regions of intimal thickening 15 days after endothelial injury, and SMC growing in culture contained a predominance of cytoplasmic actin mRNAs. Intimal SMC 60 days after endothelial injury (at which time the endothelium had fully regenerated) demonstrated a pattern of actin mRNAs similar to that of normal media. Functional mRNA measured by translation in a reticulocyte lysate showed increases in the level of alpha-actin and decreases in beta-actin in rats from 5 days to 6 weeks of age. These results suggest that during development, under pathological conditions, and in cell culture, the expression of actin isoforms in arterial SMC depends on many factors, including the amount and translation efficiency of mRNAs, and the relative stabilities of the proteins involved.

Actins↗
PubMed1985

Cytoskeletal features of rat aortic cells during development. An electron microscopic, immunohistochemical, and biochemical study.

Actin, vimentin, desmin, and tropomyosin distribution in rat aortic endothelial and smooth muscle cells has been studied during development using fetal (18 to 20 days of gestation), and 5- and 14-day-, and 5-, and 12-week-old rats. Endothelial cells of newborn animals actively replicate and contain many actin stress fibers, whereas, in adult animals, replication is minimal and actin stress fibers are rare. The actin, vimentin, desmin, and tropomyosin content of smooth muscle cells increases gradually from fetal to adult animals. The number of desmin-containing cells also increases from 13% in fetal rats to 51% in adult rats. The beta-actin isoform is predominant in fetal and newborn animals, but gradually the alpha-isoform becomes quantitatively the most important, as seen by bidimensional polyacrylamide gels. Several analogies exist between the features of developing smooth muscle and what is known for developing striated muscle cells. The evolution of cytoskeletal features from fetal to adult animals is remarkably the opposite of what takes place in: (1) rat aortic smooth muscle cells proliferating after an endothelial injury, (2) human arterial smooth muscle cells present in atheromas, and (3) actively growing rat aortic smooth muscle cells in vitro. Thus, the assumption that pathological or cultured smooth muscle cells are "dedifferentiated" is supported by our biochemical observations.

Actins↗
PubMed1984

Skin gas tensions and resistances measured by mass spectrometry in adults.

We used a dual-sampling chamber 2.5 cm in diameter, controlled by an Apple II microcomputer, to measure gas tensions in the skin of adults by mass spectrometry. The tensions of O2, CO2, N2, and water vapor and the resistances to the flows of gases through the skin were calculated from the voltages for the different gases passing through two separate membranes of different permeability. We compared the skin O2 and CO2 tensions with arterial values in 13 patients with lung disease during a standardized exercise test. The skin values followed changes in the arterial values. The regression line relating skin to arterial O2 tension had a slope of 1.05 and that for CO2 a slope of 1.51. We conclude that mass spectrometry is capable of providing measurements of gas tension from the heated skin that are similar to those found using electrochemical methods in adults.

Carbon Dioxide↗
PubMed1984

Actin expression in smooth muscle cells of rat aortic intimal thickening, human atheromatous plaque, and cultured rat aortic media.

Actin of smooth muscle cells of rat and human aortic media shows a predominance of the alpha-isoform. In experimental rat aortic intimal thickening, in human atheromatous plaque, and in cultured aortic smooth muscle cells, there is a typical switch in actin expression with a predominance of the beta-form and a noticeable amount of gamma-form. This pattern of actin expression represents a new reliable protein-chemical marker of experimental and human atheromatous smooth muscle cells.

Actins↗
PubMed1984

Cytoskeleton of rat aortic smooth muscle cells. Normal conditions and experimental intimal thickening.

The organization of actin, vimentin, and desmin in smooth muscle cells of rat aortic media and intima under normal conditions and 15 or 75 days after endothelial injury has been studied by means of electron microscopy, indirect immunofluorescence, densitometric analysis of sodium dodecyl sulfate-polyacrylamide gels, isoelectric focusing, and bidimensional gels. In the normal aortic media, practically all smooth muscle cells contain vimentin, and about 50% of them contain, in addition, desmin; upon analysis of actin isotypes by bidimensional gels, smooth muscle cells show a predominance of alpha-actin, some beta-actin, and very little gamma-actin. Fifteen days after endothelial injury, cells that have migrated into the intima contain decreased amounts of actin and desmin and increased amounts of vimentin compared with normal medial smooth muscle cells. Moreover, beta-actin becomes the predominant actin isotype and significant amounts of gamma-actin appear, whereas alpha-actin decreases. Seventy-five days after endothelial injury, regenerated endothelial cells have repaired the injury. Intimal smooth muscle cells are less numerous than 15 days after injury, and the organization of their cytoskeletal elements has reverted almost to normal conditions. At both 15 and 75 days after endothelial injury, no significant changes of cytoskeletal elements are seen in the aortic media underlying intimal thickenings.

Actins↗
PubMed1983

Cytocontractile and cytoskeletal elements in pathologic processes. Pathogenetic role and diagnostic value.

The morphologic and biochemical characterization of cytocontractile and cytoskeletal elements in a given cell or tissue appears more and more as a useful tool in the study of differentiation phenomena, the origin of various human tumors (including metastases), and the degree of cellular adaptation during physiologic and pathologic processes. Thus, the biologic features of cytocontractile and cytoskeletal elements may be of practical interest to the pathologist and may furnish important information concerning basic cell responses during disease processes.

Cell Movement↗
PubMed1981

Desmosomes and gap junctions in precarcinomatous and carcinomatous conditions of squamous epithelia. An electron microscopic and morphometrical study.

By means of morphometrical techniques we have studied the distribution of gap junctions and desmosomes in uterine epithelial cells during normal, preneoplastic and neoplastic conditions, and in skin and oral cavity during normal and neoplastic conditions. The percentage of cell surface occupied by gap junctions as well as the surface to volume ratio of these structures decreases progressively from normal to metaplastic and moderately dysplastic cervical epithelium and is practically null in more severe conditions. The percentage of cell surface occupied by desmosomes decreased significantly from normal to metaplastic epithelium; the decrease is less sharp from metaplastic epithelium to dysplastic epithelium and carcinoma in situ; it then becomes again abrupt from carcinoma in situ to invasive carcinoma. In skin and oral cavity, there is no significant change between the percentage of cell surface occupied by gap junctions as well as the surface to volume ratio of these structures in normal epithelium and basal cell carcinoma. There is a significant change between normal epithelium and squamous cell carcinoma as well as between basal cell carcinoma and squamous cell carcinoma. The percentage of cell surface occupied by desmosomes and the surface to volume ratio of desmosomes decreases significantly between normal epithelium and basal or squamous cell carcinomas as well as between basal and squamous cell carcinomas. It is probable that a lack of chemical and electronic coupling as well as a lack of mechanical adhesion play a role in determining the invasive behavior of malignant cells.

Adult↗