Overexpression of vascular permeability factor/vascular endothelial growth factor and its receptors in psoriasis.

Psoriatic skin is characterized by microvascular hyperpermeability and angioproliferation, but the mechanisms responsible are unknown. We report here that the hyperplastic epidermis of psoriatic skin expresses strikingly increased amounts of vascular permeability factor (VPF; vascular endothelial growth factor), a selective endothelial cell mitogen that enhances microvascular permeability. Moreover, two VPF receptors, kdr and flt-1, are overexpressed by papillary dermal microvascular endothelial cells. Transforming growth factor alpha (TGF-alpha), a cytokine that is also overexpressed in psoriatic epidermis, induced VPF gene expression by cultured epidermal keratinocytes. VPF secreted by TGF-alpha-stimulated keratinocytes was bioactive, as demonstrated by its mitogenic effect on dermal microvascular endothelial cells in vitro. Together, these findings suggest that TGF-alpha regulates VPF expression in psoriasis by an autocrine mechanism, leading to vascular hyperpermeability and angiogenesis. Similar mechanisms may operate in tumors and in healing skin wounds which also commonly express both VPF and TGF-alpha.

Defective jejunal and colonic salt absorption and alteredNa(+)/H (+) exchanger 3 (NHE3) activity in NHE regulatory factor 1 (NHERF1) adaptor protein-deficient mice.

We investigated the role of the Na(+)/H(+) exchanger regulatory factor 1 (NHERF1) on intestinal salt and water absorption, brush border membrane (BBM) morphology, and on the NHE3 mRNA expression, protein abundance, and transport activity in the murine intestine. NHERF1-deficient mice displayed reduced jejunal fluid absorption in vivo, as well as an attenuated in vitro Na(+) absorption in isolated jejunal and colonic, but not of ileal, mucosa. However, cAMP-mediated inhibition of both parameters remained intact. Acid-activated NHE3 transport rate was reduced in surface colonocytes, while its inhibition by cAMP and cGMP was normal. Immunodetection of NHE3 revealed normal NHE3 localization in the BBM of NHERF1 null mice, but NHE3 abundance, as measured by Western blot, was significantly reduced in isolated BBM from the small and large intestines. Furthermore, the microvilli in the proximal colon, but not in the small intestine, were significantly shorter in NHERF1 null mice. Additional knockout of PDZK1 (NHERF3), another member of the NHERF family of adaptor proteins, which binds to both NHE3 and NHERF1, further reduced basal NHE3 activity and caused complete loss of cAMP-mediated NHE3 inhibition. An activator of the exchange protein activated by cAMP (EPAC) had no effect on jejunal fluid absorption in vivo, but slightly inhibited NHE3 activity in surface colonocytes in vitro. In conclusion, NHERF1 has segment-specific effects on intestinal salt absorption, NHE3 transport rates, and NHE3 membrane abundance without affecting mRNA levels. However, unlike PDZK1, NHERF1 is not required for NHE3 regulation by cyclic nucleotides.

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.

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.

Identification of a novel gene, selectively up-regulated in human carcinomas, using the differential display technique.

Using the differential display technique, selecting for genes up-regulated in renal cell carcinoma compared with normal renal parenchyma, we isolated a novel gene, designated DD96. As determined by in situ and Northern blot hybridization studies, DD96 is expressed only in rare normal epithelial cell populations, such as the proximal tubular epithelial cells of the kidney. However, it is expressed diffusely in malignant epithelial cells of the wide majority of renal cell carcinomas. In addition, DD96 is overexpressed markedly in various human carcinomas originating from the colon, breast, and lung, as well as in a number of cell lines derived from tumors of these organs compared with normal epithelial cell populations. Furthermore, the expression of DD96 is induced in immortalized breast ductal epithelial cell lines compared with normal breast ductal epithelial cells, and, in vivo, in premalignant conditions, such as adenoma of the colon and ductal carcinoma in situ of the breast. Sequence analysis of a complete cDNA clone isolated from a human kidney cDNA library revealed that DD96 encodes for a protein of approximately Mr 13,500. These results suggest that DD96 may play a role in the early events associated with malignant transformation; however, its function remains to be determined.

Cloning of DLM-1, a novel gene that is up-regulated in activated macrophages, using RNA differential display.

Tumors interact with their environment, reprogramming host cells to induce responses such as angiogenesis, inflammation, immunity and immune suppression. To understand these processes, it is important to identify and isolate new genes whose expression is induced in host tissues in response to tumors. Ascites tumors offer an attractive model for isolating such genes, because responding host peritoneal lining tissues can be cleanly separated from tumor cells growing in suspension within the peritoneal cavity. We here report the cloning by differential display of a novel gene, DLM-1, that is highly up-regulated in the peritoneal lining tissue of mice bearing MOT ascites tumors. Mouse peritoneal macrophages, stimulated by IFN-gamma or LPS, also expressed significant amounts of DLM-1. Up-regulation of DLM-1 became evident by 4h after stimulation with IFN-gamma and was not blocked by cycloheximide, suggesting the presence of IFN responding elements in its transcription regulation region. DLM-1 RNA was detected at significant levels in normal mouse lung, intestinal epithelium, liver and thymus by Northern blot analysis. In situ hybridization of MOT and HT-29 mouse subcutaneous transplanted solid tumors revealed strong DLM-1 expression in the host reactive stromal cells, but not the tumor cells. Sequence analysis of the full-length cDNA clone revealed that it encodes a protein of approx. M(r) 44330 with multiple potential protein kinase C and casein kinase II phosphorylation sites. Our data suggest that DLM-1 plays a role in such important processes as host response in neoplasia.

Reversible expression of sm alpha-actin protein and sm alpha-actin mRNA in cloned cerebral endothelial cells.

The expression of smooth muscle (sm) alpha-actin was studied in cloned capillary cerebral endothelial cells of two phenotypes. Type I cells were cultured in medium containing 10% FCS, heparin and ECGS (or alpha-ECGF) and stained positive for a specific endothelial cell marker (Bandeiraea simplicifolia). Depletion of heparin and ECGS resulted in a smooth muscle-like appearance after 2-3 days. Cells of this phenotype, (type II) stained positive for the endothelial cell marker and for sm alpha-actin. In contrast to type I cells, type II cells expressed sm alpha-actin protein and mRNA as evidenced by Immunoblots and Northern blots. This phenotypic switch was shown to be reversible and so was the expression of sm alpha-actin.

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.

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.

Hodgkin's lymphoma of T-cell type: clonal association with a CD30+ cutaneous lymphoma.

The derivation of Reed-Sternberg cells in Hodgkin's lymphoma has been a subject of great interest. In most cases, Reed-Sternberg cells seem to be derived from germinal center B cells. In few sporadic cases, a T-cell origin has been shown. This article supports the concept of a T-cell derivation for rare cases of Hodgkin's lymphoma and provides evidence of a novel mechanism of pathogenesis from chronic inflammation in the skin.

Automated detection of the G20210A prothrombin mutation using the LCx microparticle enzyme immunoassay.

BACKGROUND: The prothrombin mutation, a G/A transition at position 20210 in the 3' untranslated region of the prothrombin gene, is associated with an increased risk of deep venous thrombosis and obstetrical complications. Several methods have been developed to detect the mutation; however, given the increased demand for this test in risk factor assessment, the development of simple and efficient screening methods has become necessary. METHODS: We have used a rapid, sensitive, and precise method developed by Abbott Laboratories to detect the prothrombin mutation. The method employs a polymerase chain reaction (PCR) amplification and the Abbot LCx microparticle enzyme immunoassay (MEIA) for detection. This method is able to detect and identify both homozygous and heterozygous genotypes. RESULTS: Two hundred ninety-six patients with a history of deep venous thrombosis, pulmonary embolism, preeclampsia, or cardiovascular disease and 163 control patients were included in this study. The prevalence of the mutation was 5.74% in the high-risk group and 3.06% in the control group. There was complete agreement between the results from the MEIA detection with those obtained using other detection methodologies, namely standard PCR and restriction fragment length polymorphism (RFLP) analysis. CONCLUSIONS: The MEIA detection method of the prothrombin mutation represents a simple, fast, and reliable alternative to standard methods of detection and is well suited for use in routine clinical laboratories. The results of our study confirm others' studies showing a greater incidence of G20210A prothrombin gene mutation in patients with an increased risk of venous thrombosis and pulmonary embolism as well as patients with cardiovascular disease and pregnant women with preeclampsia. It reinforces the necessity of including the screening for prothrombin mutation in populations at risk.

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 biology of smooth muscle in culture: implications for atherogenesis.

Smooth muscle cells are one of the most important, if not the most important component of atheromatous plaque. Smooth muscle cells from developing and regenerating arteries, as well as atheromatous plaques, show similar morphological and biochemical characteristics which differ from adult tissue. During primary culture, adult smooth muscle cells alter their morphology to resemble those of the developing, regenerating and atheromatous material. We propose that primary cell culture of smooth muscle cells provides a model for the study of smooth muscle cell changes during atheroma formation.

Desmosomes and gap-junctions in various epidermoid preneoplastic and neoplastic lesions of the cervix uteri.

The distribution of desmosomes and of gap-junctions was studied by morphometric means, at the electron microscopic level, in the following lesions of the uterine cervix: metaplasia, moderate and severe dysplasia, carcinoma in situ and invasive epidermoid carcinoma. The results were compared with normal exocervical epithelium. The proportion of the cell surface occupied by gap-junctions was decreased in all lesions; gap-junctions were statistically absent from moderate dysplasia and more advanced lesions. The number of desmosomes decreased gradually from metaplasia to invasive carcinoma, with each stage forming a discrete group. This gradual loss of intercellular structures with increasing degrees of malignancy might express dedifferentiation and could be associated with increasing cellular autonomy.

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).

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.

Modulation of gelsolin content in rat aortic smooth muscle cells during development, experimental intimal thickening and culture. An immunohistochemical and biochemical study.

Gelsolin is a Ca2+ and polyphosphoinositol-phospholipid regulated modulator of actin polymerization present in most mammalian cells and in plasma. Cytoplasmic gelsolin was first described in highly motile cells such as leukocytes, where actin polymerization is dynamic; however arterial smooth muscle cells (SMC), despite their stabilized actin bundles, express high levels of gelsolin. We have investigated gelsolin modulation in rat aortic SMC by immunohistochemistry, Western blotting and Northern hybridization using three models which are known to show modulation of actin isoform expression: development, aortic intimal thickening after experimental endothelial injury, and growth in culture. When related to the protein and mRNA content of adult aortic SMC, gelsolin is expressed about 50% in aortic SMC of five-day-old rats, 20-30% in SMC of intimal thickening 15 days after endothelial injury (when SMC are actively replicating) and in SMC growing in culture; in intimal thickening 60 days after injury (when SMC have returned to quiescence), the gelsolin content becomes similar to that of control SMC. The high level of gelsolin in smooth muscle (SM) tissues and the down regulation with proliferation and migration raises the question as to whether gelsolin in these cells has functions other than the dynamic control of actin filament length. The similar modulation patterns of gelsolin and alpha-SM actin suggest a preferential interaction between these two proteins.