MUC1-C activates BMI1 in human cancer cells.

B-cell-specific Moloney murine leukemia virus integration site 1 (BMI1) is a component of the polycomb repressive complex 1 (PRC1) complex that is overexpressed in breast and other cancers, and promotes self-renewal of cancer stem-like cells. The oncogenic mucin 1 (MUC1) C-terminal (MUC1-C) subunit is similarly overexpressed in human carcinoma cells and has been linked to their self-renewal. There is no known relationship between MUC1-C and BMI1 in cancer. The present studies demonstrate that MUC1-C drives BMI1 transcription by a MYC-dependent mechanism in breast and other cancer cells. In addition, we show that MUC1-C blocks miR-200c-mediated downregulation of BMI1 expression. The functional significance of this MUC1-C→︀BMI1 pathway is supported by the demonstration that targeting MUC1-C suppresses BMI1-induced ubiquitylation of H2A and thereby derepresses homeobox HOXC5 and HOXC13 gene expression. Notably, our results further show that MUC1-C binds directly to BMI1 and promotes occupancy of BMI1 on the CDKN2A promoter. In concert with BMI1-induced repression of the p16INK4a tumor suppressor, we found that targeting MUC1-C is associated with induction of p16INK4a expression. In support of these results, analysis of three gene expresssion data sets demonstrated highly significant correlations between MUC1-C and BMI1 in breast cancers. These findings uncover a previously unrecognized role for MUC1-C in driving BMI1 expression and in directly interacting with this stem cell factor, linking MUC1-C with function of the PRC1 in epigenetic gene silencing.

Intralobar lung sequestration with systemic coronary arterial supply.

Pulmonary sequestration is a rare anomaly. An accurate pre-operative evaluation of its vascular supply is essential for the surgeon's operative approach. We describe here an intrapulmonary sequestration with vascular arterial supply via the left circumflex and the right coronary artery. This case demonstrates that if aortography is unrevealing, then a coronary source should be considered in the preoperative search for the arterial supply to a pulmonary sequestration. Moreover, pulmonary sequestration should be listed in the differential diagnosis of aberrant coronary arteries.

Expression of cGMP-dependent protein kinase I and its substrate VASP in neointimal cells of the injured rat carotid artery.

BACKGROUND: Neointimal fibroproliferative lesions after balloon angioplasty remain a major clinical problem, frequently leading to restenosis of initially successfully dilated coronary arteries. The cGMP-dependent protein kinase type I (cGMP-PK I) and its substrate vasodilator-stimulated phosphoprotein (VASP), molecular targets of the atrial natriuretic factor (ANF) and nitric oxide (NO) signalling pathways, are likely to be involved in various aspects of vascular wall regulation and restenosis formation. METHODS: To investigate the occurrence of cGMP-PK I and VASP in neointimal cells in situ, we performed immunohistochemistry and immunoblotting experiments on denuded rat carotid arteries. RESULTS: Although the soluble cGMP-PK I showed a homogeneous distribution throughout the neointima, VASP apparently was more concentrated in smooth muscle cells (SMCs) lining the artery lumen, possibly reflecting enhanced growth factor stimulation of luminal SMCs. The membrane-associated cGMP-PK type II could not be detected in both the non-injured vessel wall and the restenotic tissue. CONCLUSION: The presence of both cGMP-PK I and VASP, major regulators of the actin cytoskeleton and cell motility, in neointimal tissue suggest that this emerging signal transduction pathway could be a target for the regulation and control of restenosis.

[Elective coronary implantation of a newly developed stent without conventional anticoagulation]. / Elektive koronare Implantation eines neu entwickelten Stents ohne klassische Antikoagulation.

OBJECTIVE: To assess in an open prospective study the angiographic and clinical results of the elective implantation of the recently developed AVE micro-stent (Applied Vascular Engineering, Santa Rosa, CA, USA), in combination with dual antiaggregation treatment. PATIENTS AND METHODS: Between January and December 1995 AVE micro-stents were implanted into 128 vessels in 121 patients (20 women, 101 men; mean age 60.7 +/- 9.5 [34-84] years) with symptomatic coronary heart disease (CHD). Indication for the primary implantation of the stent type was a complex morphology of the stenosis with unfavourable short- and long-term prognosis. The stent consists of a 4 mm long tubular highly flexible segment made of 0.008 inch wire and can be advanced even into tortuous vessels. After balloon dilatation of the stenosis the stent was advanced into the vessel wall at a pressure of 10-12 bar, followed by further dilatation at 16-18 bar. Conventional long-term anticoagulation was dispensed with, patients only receiving antiaggregation medication: 500 mg ticlopidine and 100 mg aspirin daily for 6 weeks. RESULTS: The primary success rate of stent implantation was 99% (121 of 122). Neither acute nor subacute thromboses were revealed during hospital stay nor was there any emergency bypass operation or early repeat balloon angioplasty. There were no abnormal bleedings. CONCLUSION: Stenoses which are unsuitable for conventional balloon angioplasty can be reliably treated with the AVE microstent. Optimal high-pressure dilatation in combination with dual antiaggregation treatment will prevent stent thrombosis and bleeding complications.

High expression of the focal adhesion- and microfilament-associated protein VASP in vascular smooth muscle and endothelial cells of the intact human vessel wall.

The focal adhesion and microfilament-associated protein VASP is a major substrate of both cAMP- and cGMP-dependent protein kinase in intact human platelets. The recent elucidation of the primary VASP structure and identity of VASP binding proteins suggest that VASP is an important component of focal contacts which link signal transduction pathways and elements controlling cell motility. In this study, the high expression of VASP in vascular smooth muscle and endothelial cells of human blood vessels is reported. Western blot and immunofluorescence analysis detected VASP in human heart, femoral artery and a uterine leiomyosarcoma. Within these tissues, smooth muscle cells, small capillaries and the endothelial cell layer were strongly stained by the VASP antiserum. In human heart, an overlapping cellular distribution of the cGMP-dependent protein kinase I (cGK I) and its substrate VASP was noted. Immunoelectron microscopy experiments with vascular smooth muscle cells of the vessel wall revealed that VASP is localized in close proximity to microfilaments, dense plaques and dense bodies. The data of this study and the properties of VASP as a major target of inhibitory vasoactive agents suggest that VASP is an important component which participates in the regulation of cell motility of human vessel wall cells in vivo.

Expression of type II cGMP-dependent protein kinase in rat kidney is regulated by dehydration and correlated with renin gene expression.

cGMP-based regulatory systems are vital for counteracting the renin-angiotensin system (RAS) which promotes volume expansion and high blood pressure. Natriuretic peptides and nitric oxide acting through their second messenger cGMP normally increase natriuresis and diuresis, and regulate renin release; however, the severe pathological state of cardiac heart failure is characterized by elevated levels of atrial natriuretic peptide that are no longer able to effectively oppose exaggerated RAS effects. There is presently limited information on the intracellular effectors of cGMP actions in the kidney. Recently we reported the cloning of the cDNA for type II cGMP-dependent protein kinase (cGK II), which is highly enriched in intestinal mucosa but was also detected for the first time in kidney. In the present study, cGK II was localized to juxtaglomerular (JG) cells, the ascending thin limb (ATL), and to a lesser extent the brush border of proximal tubules. An activator of renin gene expression, the angiotensin II type I receptor inhibitor, losartan, increased cGK II mRNA and protein three to fourfold in JG cells. In other experiments, water deprivation increased cGK II mRNA and protein three to fourfold in the inner medulla where both cGK II, and a kidney specific CI- channel shown by others to be regulated by dehydration, are localized in the ATL. Whereas additional data suggest that cGK I may primarily mediate cGMP-related changes in renal hemodynamics, cGK II may regulate renin release and ATL ion transport.

Endogenous expression of type II cGMP-dependent protein kinase mRNA and protein in rat intestine. Implications for cystic fibrosis transmembrane conductance regulator.

Certain pathogenic bacteria produce a family of heat stable enterotoxins (STa) which activate intestinal guanylyl cyclases, increase cGMP, and elicit life-threatening secretory diarrhea. The intracellular effector of cGMP actions has not been clarified. Recently we cloned the cDNA for a rat intestinal type II cGMP dependent protein kinase (cGK II) which is highly enriched in intestinal mucosa. Here we show that cGK II mRNA and protein are restricted to the intestinal segments from the duodenum to the proximal colon, with the highest amounts of cGK II protein in duodenum and jejunum. cGK II mRNA and protein decreased along the villus to crypt axis in the small intestine, whereas substantial amounts of both were found in the crypts of cecum. In intestinal epithelia, cGK II was specifically localized in the apical membrane, a major site of ion transport regulation. In contrast to cGK II, cGK I was localized in smooth muscle cells of the villus lamina propria. Short circuit current (ISC), a measure of Cl- secretion, was increased to a similar extent by STa and by 8-Br-cGMP, a selective activator of cGK, except in distal colon and in monolayers of T84 human colon carcinoma cells in which cGK II was not detected. In human and mouse intestine, the cyclic nucleotide-regulated Cl- conductance can be exclusively accounted for by the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. Viewed collectively, the data suggest that cGK II is the mediator of STa and cGMP effects on Cl- transport in intestinal-epithelia.

Platelet-vessel wall interactions, focal adhesions, and the mechanism of action of endothelial factors.

Endothelial cells produce a variety of vasoactive substances including prostacyclin (PGI2) and endothelium-derived relaxing factor (EDRF/NO) which are potent inhibitors of platelet adhesion/aggregation and vascular smooth muscle cell contraction/proliferation. PGI2 and EDRF elevate cAMP or cGMP, respectively, in vascular cells and other targets. The intracellular effects of cAMP and cGMP in vascular smooth muscle cells and platelets are primarily mediated by the family of cAMP- and cGMP-dependent protein kinases and their substrates. Important effector systems include enzymes, channels and regulatory proteins responsible for the regulation of intracellular Ca++. Other evidence suggests that VASP, a focal adhesion protein phosphorylated in platelets and smooth muscle cells in response to PGI2 and EDRF, is important for the regulation of integrins and cell-matrix interactions.

Cloning, expression, and in situ localization of rat intestinal cGMP-dependent protein kinase II.

The cDNA for a membrane-associated cGMP-dependent protein kinase (cGK II) was cloned from rat intestine using reverse transcriptase PCR and oligonucleotide primers encoding two conserved motifs of known cGMP-dependent protein kinases and subsequently by screening a rat intestine cDNA library. A full-length clone encodes a protein of 761 amino acids with an estimated size of 87 kDa. Sequences of eight peptides from purified pig intestinal mucosa cGK II were found in the derived amino acid sequence of this clone, identifying it as rat intestinal cGK II. Phylogenetic analysis showed that rat intestinal cGK II is less related to mammalian cGK I than to the Drosophila DG1 gene product and most closely related to a recently cloned mouse brain CGKII isoform. Like several other cGK sequences, that of cGK II contained a leucine/isoleucine heptad repeat motif that has been implicated in dimer formation in cGK I. Expression of cGK II cDNA in HEK 293 cells followed by subcellular fractionation revealed cGK II localization in the cell particulate fraction, consistent with the membrane association of endogenous rat cGK II. On Northern blots, the major cGK II poly(A) RNA form was 4.8 kb, with minor forms of 6.2 and 3.1 kb. The cGK II RNA was highly expressed in rat intestinal mucosa and was 20 times less abundant in rat brain and kidney. The localization of endogenous cGK II RNA in rat small intestine was shown by in situ hybridization to be in villous epithelial cells and to some extent in crypt cells.

In situ localization of transforming growth factor beta 1 in porcine heart: enhanced expression after chronic coronary artery constriction.

We investigated the expression of transforming growth factor beta 1 (TGF-beta 1), a polypeptide differentiation factor probably associated with angiogenic properties in chronically hypoperfused heart tissue. A slowly swelling ameroid constrictor was implanted around the coronary circumflex artery (CX) of young domestic pigs. Two to three weeks after, significant CX stenosis of more than 90% and coronary collateralization could be demonstrated angiographically. The CX dependent experimental myocardial tissue (E) was investigated, with the LAD dependent area of the same pig serving as a control (C). We found significantly enhanced TGF-beta 1 mRNA expression by northern blot hybridization in the experimental myocardium (E) of those pigs with demonstrable coronary collaterals in the absence of a major myocardial infarction. The presence of TGF-beta 1 protein could be demonstrated quantitatively in extracts of the experimental and the control area by immunoblot analysis. By in situ techniques, TGF-beta 1 mRNA and protein could be localized predominantly in cardiac myocytes. We conclude that one adaptive mechanism of the pig heart in chronic coronary artery constriction is the enhanced expression of TGF-beta 1. Cardiac myocytes are a major source of TGF-beta 1. The observed coronary collateralization could be mediated-at least in part-by the angiogenic properties of TGF-beta 1.

Molecular biologic concepts of coronary anastomoses.

The discovery that collateral development after progressive coronary stenosis proceeds by means of DNA synthesis, mitosis and proliferation of endothelial and smooth muscle cells in preformed small interconnecting arterioles (canine heart) and capillaries (porcine heart) has stimulated research into the molecular mechanisms of vascular growth. Growth is tightly controlled under physiologic conditions, and several factors must act in concert to overcome control. Because the result of growth is a much larger orderly structure of complex design, we expect the existence of a genetic blueprint for its construction. Peptide growth factors have recently been isolated from a variety of organs, including the heart. We have provided experimental evidence that the heparin-binding growth factor beta-ECGF shows an increased transcription in growing pig collateral vessels. Because the chain of events probably originates in the ischemic cardiac myocyte, it appears logical to search there for the initiating factor. In addition to local production, growth factors can also be transported into ischemic myocardium by blood-borne cells. Monocytes adhere to altered endothelium in a potentially ischemic region and start to produce growth factors in situ. Platelets are rich sources of transforming growth factor-beta (TGF-beta), platelet-derived endothelial cell growth factor (PDECGF) and platelet-derived growth factor (PDGF), all of which are known angiogenic factors or mitogens.