uPARAP expression during murine lung development

Lung remodeling requires active collagen deposition and degradation. Urokinase plasminogen activator receptor-associated protein (uPARAP), or Endo 180, is a cell-surface receptor for collagens, which leads to collagen internalization and degradation. Thus, uPARAP-mediated collagen degradation is an additional pathway for matrix remodeling in addition to matrix remodeling mediated by matrix metalloproteinases and cathepsins. Using immunohistochemistry, we demonstrate extensive uPARAP expression in the mesenchyme throughout murine lung development. By immunofluorescence, we demonstrate significant overlap of uPARAP expression with collagen IV expression, but minimal overlap with collagen I expression in the developing murine lung. Finally, we compared lung development between wild-type and uPARAP(-/-) mice, and found no significant histologic differences, indicating the presence of alternative collagen degradation pathways during murine lung development.     

Nitric oxide-modulated marker gene expression of signal transduction pathways in lung endothelial cells

Nitric oxide (NO) is a signal molecule involved in regulation of physiological and pathophysiological functions of the vascular endothelium such as apoptosis. We examined whether NO-modulates marker gene expression of signal transduction pathways in cultured pulmonary artery endothelial cell (PAEC). Cells were exposed to a NO donor, 1 mM NOC-18, for 0.5, 5, and 24 h, thereafter, expression levels of 96 marker genes associated with 18 signal transduction pathways were assessed using a signal transduction pathway-finder microarray analysis system. NO modulation of apoptotic pathways and nuclear factor (NF) microarray were further analyzed. Gene array analyses revealed that 17 genes in 13 signal pathways were up- or down-regulated in cells exposed to NO, four of which were significantly altered by NO and are associated with apoptotic pathways. Apoptotic pathways resulted in identification of 11 genes in this group. Nuclear factor microarray studies demonstrated that NO-modulated expression of these signal transduction genes was associated with regulation of NF-binding activities. Gel shift analysis verified the effects of NO on DNA-binding activity of NF. These results demonstrated that NO signaling modulates at least 13 signal transduction pathways including apoptosis-related families in PAEC.     

Implications of post-pneumonectomy compensatory lung growth in pulmonary physiology and disease

In a number of species, partial pneumonectomy initiates hormonally regulated compensatory growth of the remaining lung lobes that restores normal mass, structure and function. Compensation is qualitatively similar across species, but differs with gender, age and hormonal status. Although the biology of response is best characterized in rats, dogs have proven valuable in defining post-operative physiological adaptations. Most recently, mice were recognized to offer unique opportunities to explore the genetic basis of the response, as well as to evaluate associated detrimental effects of pathophysiological significance in animals exposed to carcinogens. The pneumonectomy model thus offers powerful insight concerning adaptive organ growth.     

Osteoblastic gene expression during adipogenesis in hematopoietic supporting murine bone marrow stromal cells

A growing body of data suggests that the bone marrow stroma contains a population of pluripotent cells capable of differentiating into adipocytes, osteoblasts, and lymphohematopoietic supporting cells. In this work, the murine stromal cell lines BMS2 and +/+ 2.4 have been examined as preadipocytes and adipocytes for evidence of osteoblastic gene expression. Adipocyte differentiation has been quantitated using fluorescence activated cell sorting. Within 7–10 days of adipocyte induction by treatment with glucocorticoids, indomethacin, and methylisobutylxanthine, between 40% to 50% of the cells contain lipid vacuoles and exhibit a characteristic adipocyte morphology. Based on immunocytochemistry, both the adipocytes and preadipocytes express a number of osteoblastic markers; these include alkaline phosphatase, osteopontin, collagen (I, III), bone sialoprotein II, and fibronectin. Based on biochemical assays, the level of alkaline phosphatase expression is not significantly different between preadipocyte and adipocyte cells. However, unlike rat cell lines, dexamethasone exposure causes a dose-dependent decrease in enzyme activity. The steady-state mRNA levels of the osteoblast associated genes varies during the process of adiopogenesis. The relative level of collagen I and collagen III mRNA is lower in adipocyte-induced cells when compared to the uninduced controls. Osteocalcin mRNA is detected in preadipocytes but absent in adipocytes. These data indicate that osteoblastic gene expression is detected in cells capable of undergoing adipocyte differentiation, consistent with the hypothesis that these cell lineages are interrelated. © 1993 Wiley-Liss, Inc.     

Angiogenesis activators and inhibitors differentially regulate caveolin-1 expression and caveolae formation in vascular endothelial cells. Angiogenesis inhibitors block vascular endothelial growth factor-induced down-regulation of caveolin-1.

Angiogenesis is the process by which new blood vessels are formed via proliferation of vascular endothelial cells. A variety of angiogenesis inhibitors that antagonize the effects of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) have recently been identified. However, the mechanism by which these diverse angiogenesis inhibitors exert their common effects remains largely unknown. Caveolin-1 and -2 are known to be highly expressed in vascular endothelial cells both in vitro and in vivo. Here, we examine the potential role of caveolins in the angiogenic response. For this purpose, we used the well established human umbilical vein endothelial cell line, ECV 304. Treatment of ECV 304 cells with known angiogenic growth factors (VEGF, bFGF, or hepatocyte growth factor/scatter factor), resulted in a dramatic reduction in the expression of caveolin-1. This down-regulation event was selective for caveolin-1, as caveolin-2 levels remained constant under these conditions of growth factor stimulation. VEGF-induced down-regulation of caveolin-1 expression also resulted in the morphological loss of cell surface caveolae organelles as seen by transmission electron microscopy. A variety of well characterized angiogenesis inhibitors (including angiostatin, fumagillin, 2-methoxy estradiol, transforming growth factor-beta, and thalidomide) effectively blocked VEGF-induced down-regulation of caveolin-1 as seen by immunoblotting and immunofluorescence microscopy. However, treatment with angiogenesis inhibitors alone did not significantly affect the expression of caveolin-1. PD98059, a specific inhibitor of mitogen-activated protein kinase and a known angiogenesis inhibitor, also blocked the observed VEGF-induced down-regulation of caveolin-1. Furthermore, we show that caveolin-1 can function as a negative regulator of VEGF-R (KDR) signal transduction in vivo. Thus, down-regulation of caveolin-1 may be an important step along the pathway toward endothelial cell proliferation.     

Transcriptional regulation of basic fibroblast growth factor gene expression in capillary endothelial cells.

The growth of capillary endothelial cells (BCE) is an important regulatory step in the formation of capillary blood vessels. In vivo, the proliferation of these cells is stringently controlled. In vitro they can be stimulated by polypeptide growth factors, such as acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF). Since bFGF is synthesized and stored by vascular endothelial cells, this mitogen may play an important role in an autocrine growth regulation during angiogenesis. Here, evidence is presented for induction of the mRNA of bFGF by bFGF itself. A similar increase of bFGF mRNA was observed in response to thrombin and after treatment with phorbol ester. These results suggest that an autocrine loop may exist that may serve to modulate the mitogenic response in BCE under various physiological conditions, (e.g., wound healing and new capillary formation).     

Vascular endothelial growth factor expression and regulation of murine collagen-induced arthritis

We have examined the expression and function of the angiogenic factor, vascular endothelial growth factor (VEGF) during the evolution of type II collagen-induced arthritis (CIA). Biologically active VEGF was expressed along a time course that paralleled the expression of two specific VEGF receptors, Flk-1 and Flt-1, and the progression of joint disease. Moreover, levels of VEGF expression correlated with the degree of neovascularization, as defined by vWF levels, and arthritis severity. Macrophage- and fibroblast-like cells, which infiltrated inflamed sites and were then activated by other inflammatory mediators, are probably important sources of VEGF and may thus regulate angiogenesis during the development of CIA. Administration of anti-VEGF antiserum to CIA mice before the onset of arthritis delayed the onset, reduced the severity, and diminished the vWF content of arthritic joints. By contrast, administration of anti-VEGF antiserum after the onset of the disease had no effect on the progression or ultimate severity of the arthritis. These data suggest that VEGF plays a crucial role during an early stage of arthritis development, affecting both neovascularization and the progression of experimentally induced synovitis.     

Thrombin stimulates c-sis gene expression in microvascular endothelial cells

We have determined whether expression of the c-sis gene product, platelet-derived growth factor (PDGF), is regulated in cultured renal microvascular endothelial cells by factors to which vascular endothelial cells may be exposed at sites of perivascular cellular proliferation. Thrombin exposure increased endothelial cell levels of c-sis message by 3-5-fold over a time course that peaked at 4 h after exposure. Similarly, thrombin-exposed microvascular endothelial cells released increased amounts of PDGF activity into their media. The thrombin effect was not mediated through the proteolytic activity of thrombin, as proteolytically inactive thrombin stimulated the c-sis expression as well as native thrombin. This stimulation was mimicked by exposure of cells to biologically active phorbol esters, suggesting that thrombin action may be mediated through activation of kinase C (Ca2+/phospholipid-dependent enzyme). Thus, thrombin regulates the expression and release of PDGF activity from endothelial cells in culture and may act in vivo to stimulate mitogen release from endothelial cells, thereby inducing proliferation of perivascular cells.     

The dynamics of gene expression in human lung microvascular endothelial cells after stimulation with inflammatory cytokines

Vascular endothelium plays an essential role in the pathogenesis of vasoocclusion. The changes in the endothelial cell function can be triggered by changes in gene expression caused by interaction with cytokines and blood cells. Using cDNA arrays, we have recently reported complex patterns of gene expression after stimulation of endothelial cells with TNFalpha and IL-1beta. Better understanding of the time course of gene expression changes, their concentration dependence and reversibility after withdrawal of the offending cytokine is essential for successful prevention and therapy of vasoocclusion. Here we present a detailed study of the concentration dependence and time course of gene expression in endothelial cells after their exposure to TNFalpha and IL-1beta. We focus on the adhesion molecules (VCAM-1, ICAM-1, E-selectin) and cytokines (IL-6, GCP-2, MCP-1) that are likely to contribute to vasoocclusion. We report differences in the time course and intensity of their expression and in their response to TNFalpha and IL-1beta stimulation. We demonstrate that expression of the studied genes is upregulated by low TNFalpha concentrations that better reflect the TNFalpha levels detected in the plasma of patients developing vasoocclusion. These results help to understand the changes in the endothelium and to design rational prevention and therapy of vasoocclusion.