Differential expression of TGF beta 1, beta 2 and beta 3 genes during mouse embryogenesis

We have examined by Northern analysis and in situ hybridisation the expression of TGF beta 1, beta 2 and beta 3 during mouse embryogenesis. TGF beta 1 is expressed predominantly in the mesodermal components of the embryo e.g. the hematopoietic cells of both fetal liver and the hemopoietic islands of the yolk sac, the mesenchymal tissues of several internal organs and in ossifying bone tissues. The strongest TGF beta 2 signals were found in early facial mesenchyme and in some endodermal and ectodermal epithelial cell layers e.g., lung and cochlea epithelia. TGF beta 3 was strongest in prevertebral tissue, in some mesothelia and in lung epithelia. All three isoforms were expressed in bone tissues but showed distinct patterns of expression both spatially and temporally. In the root sheath of the whisker follicle, TGF beta 1, beta 2 and beta 3 were expressed simultaneously. We discuss the implication of these results in regard to known regulatory elements of the TGF beta genes and their receptors.     

Domain V peptides inhibit beta2-glycoprotein I-mediated mesenteric ischemia/reperfusion-induced tissue damage and inflammation

Reperfusion of ischemic tissue induces significant tissue damage in multiple conditions, including myocardial infarctions, stroke, and transplantation. Although not as common, the mortality rate of mesenteric ischemia/reperfusion (IR) remains >70%. Although complement and naturally occurring Abs are known to mediate significant damage during IR, the target Ags are intracellular molecules. We investigated the role of the serum protein, β2-glycoprotein I as an initiating Ag for Ab recognition and β2-glycoprotein I (β2-GPI) peptides as a therapeutic for mesenteric IR. The time course of β2-GPI binding to the tissue indicated binding and complement activation within 15 min postreperfusion. Treatment of wild-type mice with peptides corresponding to the lipid binding domain V of β2-GPI blocked intestinal injury and inflammation, including cellular influx and cytokine and eicosanoid production. The optimal therapeutic peptide (peptide 296) contained the lysine-rich region of domain V. In addition, damage and most inflammation were also blocked by peptide 305, which overlaps with peptide 296 but does not contain the lysine-rich, phospholipid-binding region. Importantly, peptide 296 retained efficacy after replacement of cysteine residues with serine. In addition, infusion of wild-type serum containing reduced levels of anti-β2-GPI Abs into Rag-1(-/-) mice prevented IR-induced intestinal damage and inflammation. Taken together, these data suggest that the serum protein β2-GPI initiates the IR-induced intestinal damage and inflammatory response and as such is a critical therapeutic target for IR-induced damage and inflammation.     

Time course of ischemia/reperfusion-induced oxidative modification of neural proteins in rat forebrain

Time course of oxidative modification of forebrain neural proteins was investigated in the rat model of global and partial cerebral ischemia/reperfusion. Animals were subjected to 4-vessel occlusion for 15 min (global ischemia). After the end of ischemia and at different reperfusion times (2, 24 and 48 h), lipoperoxidation-dependent and direct oxidative modification neural protein markers were measured in the forebrain total membrane fraction (tissue homogenate). Ischemia itself causes significant changes only in levels of tryptophan and bityrosine fluorescence when compared to controls. All tested parameters of protein modification altered significantly and were maximal at later reperfusion stage. Content of carbonyl group in re-flow period steadily increased and culminated at 48 h of reperfusion. The highest increase in the fluorescence of bityrosines was detected after 24 h of reperfusion and was statistically significant to both sham operated and ischemic groups. The changes in fluorescence intensity of tryptophan decreased during a reperfusion time dependent manner. Formation of lysine conjugates with lipoperoxidation end-products significantly increased only at later stages of reperfusion. Total forebrain membranes from animals subjected to 3-vessel occlusion model to 15 min (partial ischemia) show no altered content of oxidatively modified proteins compared to controls. Restoration of blood flow for 24 h significantly decreased only fluorescence of aromatic tryptophan. Partial forebrain ischemia/reperfusion resulted in no detectable significant changes in oxidative products formation in extracerebral tissues (liver and kidney) homogenates. Our results suggest that global ischemia/reperfusion initiates both the lipoperoxidation-dependent and direct oxidative modifications of neural proteins. The findings support the view that spatial and temporal injury at later stages of ischemic insult at least partially involves oxidative stress-induced amino acid modification. The results might have important implications for the prospective post-ischemic antioxidant therapy.     

Developmental expression of the TGF beta s in the mouse cochlea.

Mice with targeted disruption of the TGF beta 2 gene display defects in epithelial-mesenchymal tissue interactions in several tissues including the developing cochlea. Specifically, the region of the spiral limbus and the overlying interdental cells, structures putatively involved in endolymphatic fluid homeostasis, display morphogenetic abnormalities. These findings prompted us to explore the pre-natal and post-natal expression of all three mammalian TGF beta genes in the developing mouse inner ear. TGF beta 2 mRNA expression was identified throughout the cochlear epithelium at all of the developmental stages examined. TGF beta 3 mRNA expression was identified in the mesenchymal tissues of the cochlea surrounding the otic epithelium. We found no evidence for compensation by the other two TGF beta isoforms in the cochleas of the TGF beta 2 mutants.     

Effect of ischemic preconditioning on pancreatic regeneration and pancreatic expression of vascular endothelial growth factor and platelet-derived growth factor-A in ischemia/reperfusion-induced pancreatitis.

Ischemic preconditioning has been shown to protect several organs from ischemia/reperfusion-induced injury. In the pancreas, protective effect of ischemic preconditioning has been shown against pancreatitis evoked by ischemia/reperfusion, as well as by caerulein. However, the effect of ischemic preconditioning on the course of acute pancreatic is unclear. The aim of our study was to evaluate the influence of ischemic preconditioning on pancreatic regeneration and pancreatic presence of platelet-derived growth factor-A (PDGF-A) and vascular endothelial growth factor (VEGF) in the course of ischemia/reperfusion-induced pancreatitis. METHODS: In male Wistar rats, ischemic preconditioning of the pancreas was performed by short-term clamping of celiac artery (twice for 5 min with 5 min interval). Acute pancreatitis was induced by clamping of inferior splenic artery for 30 min followed by reperfusion. Rats were sacrificed 1, 5, 12 h or 1, 2, 3, 5, 7, 9 and 21 days after the start of reperfusion. Severity of acute pancreatitis and pancreatic regeneration were determined by biochemical and morphological examination, expression of growth factors was determined by immunohistochemical analysis. RESULTS: In ischemia/reperfusion-induced pancreatitis, the pancreatic damage reached the maximal range between the first and second day of reperfusion, and was followed by subsequent pancreatic regeneration. Ischemic preconditioning alone caused mild passing pancreatic damage and an increase in plasma concentration of pro-inflammatory interleukin-1 and anti-inflammatory interleukin-10. Ischemic preconditioning applied before ischemia/reperfusion-induced pancreatitis reduced morphological and biochemical signs of the pancreatitis-evoked pancreatic damage and accelerated pancreatic regeneration. This effect was associated with improvement of pancreatic blood flow. Ischemic preconditioning, ischemia/reperfusion-induced pancreatitis and their combination increased the presence of VEGF in acinar and islet cells, and immunostaining for PDGF-A in blood vessels. This effect was maximally pronounced after combination of ischemic preconditioning plus pancreatitis and occurred earlier than after pancreatitis alone. CONCLUSIONS: Ischemic preconditioning reduces pancreatic damage and accelerates pancreatic healing in the course of ischemia/reperfusion-induced pancreatitis. This effect is associated with the increase in plasma concentration of anti-inflammatory interleukin-10, improvement of pancreatic blood flow and alteration of pancreatic immunohistochemical expression of PDGF-A and VEGF.     

Deterioration of ischemia/reperfusion-induced acute renal failure in SOD1-deficient mice

Reactive oxygen species (ROS) are likely candidates for involvement in ischemia/reperfusion-induced acute renal failure (ARF). In this study, the issue of whether superoxide dismutase (SOD1)-deficiency exacerbates the ischemia/reperfusion-induced ARF was examined. At two weeks after a right nephrectomy of mice, the left renal vessels were clipped to induce renal ischemia and were then released after 45 min. The severe renal damage observed at one day was partially recovered at seven days after the induction of ischemia. SOD1^- / -  mice suffer from severe ARF compared with SOD1^+/ -  and SOD1^+/+ mice. The damage was more evident in aged animals (24-28 week old) than younger ones (10-12 week old). The expression of major antioxidative and redox enzymes, except for CuZnSOD, were substantially unchanged. Thus, the increased ARF in SOD1^- / -  mice appears to be mainly attributable to a deficiency in CuZnSOD. These data support the view that ROS are exacerbating factors in ischemia/reperfusion-induced ARF.     

Deterioration of ischemia/reperfusion-induced acute renal failure in SOD1-deficient mice

Reactive oxygen species (ROS) are likely candidates for involvement in ischemia/reperfusion-induced acute renal failure (ARF). In this study, the issue of whether superoxide dismutase (SOD1)-deficiency exacerbates the ischemia/reperfusion-induced ARF was examined. At two weeks after a right nephrectomy of mice, the left renal vessels were clipped to induce renal ischemia and were then released after 45 min. The severe renal damage observed at one day was partially recovered at seven days after the induction of ischemia. SOD1^{? / ?} mice suffer from severe ARF compared with SOD1^{+/ ?} and SOD1^+/+ mice. The damage was more evident in aged animals (24–28 week old) than younger ones (10–12 week old). The expression of major antioxidative and redox enzymes, except for CuZnSOD, were substantially unchanged. Thus, the increased ARF in SOD1^{? / ?} mice appears to be mainly attributable to a deficiency in CuZnSOD. These data support the view that ROS are exacerbating factors in ischemia/reperfusion-induced ARF.     

Immunohistochemical demonstration of villin in the normal human pancreas and in chronic pancreatitis

Summary Human pancreatic tissue was investigated by immunohistochemistry using a polyclonal antibody against the actin binding protein villin, which participates in the formation of actin filament bundles in the microvilli. In cells of the different parts of the pancreatic duct system as well as in the acinar cells villin immunoreactivity was located mainly at the apical cell surface. This was confirmed by the ultrastructural demonstration of microvilli on the surface of duct and acinar cells, which exhibited the typical actin bundles. In chronic pancreatitis the staining for villin in duct-like structures of degenerative pancreatic tissue was irregular or even absent. This correlated with the electron microscopic observation of duct-like structures known as tubular complexes composed of cells devoid of microvilli at the apical cell surface. At the light microscopical level degenerative structures without lumen and of unknown origin showed a strong staining for villin at their basal cell surface.     

Immunohistochemical demonstration of villin in the normal human pancreas and in chronic pancreatitis

Human pancreatic tissue was investigated by immunohistochemistry using a polyclonal antibody against the actin binding protein villin, which participates in the formation of actin filament bundles in the microvilli. In cells of the different parts of the pancreatic duct system as well as in the acinar cells villin immunoreactivity was located mainly at the apical cell surface. This was confirmed by the ultrastructural demonstration of microvilli on the surface of duct and acinar cells, which exhibited the typical actin bundles. In chronic pancreatitis the staining for villin in duct-like structures of degenerative pancreatic tissue was irregular or even absent. This correlated with the electron microscopic observation of duct-like structures known as tubular complexes composed of cells devoid of microvilli at the apical cell surface. At the light microscopical level degenerative structures without lumen and of unknown origin showed a strong staining for villin at their basal cell surface.     

Preconditioning decreases ischemia/reperfusion-induced release and activation of matrix metalloproteinase-2

Release and activation of matrix metalloproteinases (MMPs) significantly contribute to myocardial stunning injury immediately after ischemia and reperfusion, however, their role in preconditioning remains unknown. We therefore examined the effects of preconditioning and subsequent ischemia/reperfusion on MMP activity in isolated rat hearts. Hearts were subjected to a preconditioning protocol (three consecutive 5-min periods of global ischemia interspersed with 5 min of reperfusion) followed by 30 min ischemia and 5 min reperfusion. To measure MMP release, coronary effluent was collected: (a) during aerobic perfusion, (b) in reperfusion following each preconditioning ischemia, and (c) during the final reperfusion following test ischemia. MMP-2 activities could be detected by gelatin zymography in the ventricles and coronary effluent samples from the perfused hearts. The levels of MMP-2 activity in the effluent were markedly increased in effluent following test ischemia from control hearts without preconditioning. This was accompanied by a decrease in corresponding tissue MMP activities. Preconditioning significantly decreased the MMP-2 activity in the coronary effluent following test ischemia/reperfusion and preserved the MMP-2 protein content and activity in the myocardium. Our results demonstrate that classic preconditioning inhibits ischemia/reperfusion induced release and activation of MMP-2. These results suggest that preconditioning may exert part of its cardioprotective effects through the reduction of MMP-2 release.     

FGF-2, IL-1beta and TGF-beta regulate fibroblast expression of S100A8

Growth factors, including fibroblast growth factor-2 (FGF-2) and transforming growth factor-beta (TGF-beta) regulate fibroblast function, differentiation and proliferation. S100A8 and S100A9 are members of the S100 family of Ca2+-binding proteins and are now accepted as markers of inflammation. They are expressed by keratinocytes and inflammatory cells in human/murine wounds and by appropriately activated macrophages, endothelial cells, epithelial cells and keratinocytes in vitro. In this study, regulation and expression of S100A8 and S100A9 were examined in fibroblasts. Endotoxin (LPS), interferon gamma (IFNgamma), tumour-necrosis factor (TNF) and TGF-beta did not induce the S100A8 gene in murine fibroblasts whereas FGF-2 induced mRNA maximally after 12 h. The FGF-2 response was strongly enhanced and prolonged by heparin. Interleukin-1beta (IL-1beta) alone, or in synergy with FGF-2/heparin strongly induced the gene in 3T3 fibroblasts. S100A9 mRNA was not induced under any condition. Induction of S100A8 in the absence of S100A9 was confirmed in primary fibroblasts. S100A8 mRNA induction by FGF-2 and IL-1beta was partially dependent on the mitogen-activated-protein-kinase pathway and dependent on new protein synthesis. FGF-2-responsive elements were distinct from the IL-1beta-responsive elements in the S100A8 gene promoter. FGF-2-/heparin-induced, but not IL-1beta-induced responses were significantly suppressed by TGF-beta, possibly mediated by decreased mRNA stability. S100A8 in activated fibroblasts was mainly intracytoplasmic. Rat dermal wounds contained numerous S100A8-positive fibroblast-like cells 2 and 4 days post injury; numbers declined by 7 days. Up-regulation of S100A8 by FGF-2/IL-1beta, down-regulation by TGF-beta, and its time-dependent expression in wound fibroblasts suggest a role in fibroblast differentiation at sites of inflammation and repair.     

Cyclic mechanical stretch induces VEGF and FGF-2 expression in pulmonary vascular smooth muscle cells

Vascular endothelial growth factor (VEGF) and basic (b) fibroblast growth factor (FGF-2/bFGF) are involved in vascular development and angiogenesis. Pulmonary artery smooth muscle cells express VEGF and FGF-2 and are subjected to mechanical forces during pulsatile blood flow. The effect of stretch on growth factor expression in these cells is not well characterized. We investigated the effect of cyclic stretch on the expression of VEGF and FGF-2 in ovine pulmonary artery smooth muscle cells. Primary confluent cells from 6-wk-old lambs were cultured on flexible silicon membranes and subjected to cyclic biaxial stretch (1 Hz; 5-25% stretch; 4-48 h). Nonstretched cells served as controls. Expression of VEGF and FGF-2 was determined by Northern blot analysis. Cyclic stretch induced expression of both VEGF and FGF-2 mRNA in a time- and amplitude-dependent manner. Maximum expression was found at 24 h and 15% stretch (VEGF: 1.8-fold; FGF-2: 1.9-fold). These results demonstrate that mechanical stretch regulates VEGF and FGF-2 gene expression, which could play a role in pulmonary vascular development or in postnatal pulmonary artery function or disease.     

Oxygen radical-dependent expression of CXC chemokines regulate ischemia/reperfusion-induced leukocyte adhesion in the mouse colon

Activation and accumulation of leukocytes constitute a rate-limiting step in ischemia/reperfusion (I/R)-induced tissue injury. The signalling mechanisms, however, that regulate leukocyte rolling and adhesion in the colonic microcirculation are not known. The objective of the study was to define the role of CXC chemokines (MIP-2 and KC) in I/R-induced leukocyte-endothelial cell interactions in the mouse colon. In C57/B16 mice, colonic ischemia was induced by clamping the superior mesenteric artery for 30 min and leukocyte rolling and stationary adhesion were examined in venules after 120 and 240 min of reperfusion. I/R provoked a clear-cut increase in leukocyte rolling and adhesion in colonic venules. Both MIP-2 and KC were upregulated at the gene and protein level in the reperfused colon. Immunoneutralization of MIP-2 and KC by monoclonal antibodies reduced reperfusion-induced firm adhesion of leukocytes by 73% and 75%, respectively. Interestingly, combined inhibition of MIP-2 and KC additionally decreased leukocyte rolling by 79%, but did not further reduce the number of firmly adherent leukocytes. To study the role of oxygen free radicals (OFRs) in the regulation of CXC chemokine expression, additional animals were pretreated with the xanthine-oxidase inhibitor allopurinol. In fact, allopurinol treatment reduced the colonic levels of MIP-2 and KC by 62% and 64%, respectively. This study elucidates important interactions between OFRs and chemokines in the I/R-induced leukocyte response in the mouse colon. Moreover, our data demonstrate that CXC chemokines play a fundamental role in colonic I/R and that functional interference with CXC chemokines may protect against pathological inflammation in the colon.     

Identification of the MAP2- and P75-binding domain in the regulatory subunit (RII beta) of type II cAMP-dependent protein kinase. Cloning and expression of the cDNA for bovine brain RII beta

cDNA clones coding for the regulatory subunit (RII beta) of type II cAMP-dependent protein kinase were isolated from a bovine brain cDNA expression library in lambda gt11. The cDNA codes for a protein of 418 amino acids which is 98% homologous to the rat and human RII beta proteins. A series of expression vectors coding for truncated RII beta proteins were constructed in pATH plasmids and fusion proteins were expressed in Escherichia coli. Polyclonal and monoclonal antibodies made against purified bovine brain RII were immunoreactive with the fusion proteins on Western blots. The expressed RII beta-fusion proteins were used in overlay assays to identify the region in RII beta which binds to microtubule-associated protein 2 (MAP2) and to the 75,000-dalton calmodulin-binding protein (P75) (Sarkar, D., Erlichman, J., and Rubin, C.S. (1984) J. Biol. Chem. 259, 9844-9846) in bovine brain. Fusion protein containing amino acids 1-50 of the RII beta NH2 terminus (RII beta(1-50)] bound to both MAP2 and P75 immobilized on nitrocellulose filters. A pATH11-directed fusion protein containing the 31 amino acid RII-binding site of the human MAP2 protein (MAP2(31)) (Rubino, H.M., Dammerman, M., Shafit-Zagardo, B., and Erlichman, J. (1989) Neuron 3, 631-638) also bound RII beta-fusion proteins containing RII beta amino acids 1-50. Three fusion proteins, RII beta(1-25), RII beta(25-96), and RII beta(1-265,25-96 deleted) did not bind to MAP2(31) nor P75. The results showed that the binding domain for MAP2 and P75 was located within the NH2-terminal 50 amino acids of RII beta. Preincubation of bovine heart protein kinase II alpha and RII beta(1-50) with MAP2(31) prevented their binding to both P75 and MAP2(31) that were immobilized on nitrocellulose, suggesting that the binding sites for MAP2 and P75 are located near each other or that the same site on RII was binding to both proteins.     

Growth stimulation and differential regulation of transforming growth factor-beta 1 (TGF beta 1), TGF beta 2, and TGF beta 3 messenger RNA levels by norethindrone in MCF-7 human breast cancer cells.

Transforming growth factor-beta (TGF beta) is a potent growth inhibitor in most epithelial cells. We evaluated the effects of norethindrone (which in combination with estrogen is commonly used in oral contraceptives) and other progestins [medioxyprogesterone acetate (MPA) and R5020, which are not used in oral contraceptives] on cell growth and the expression of TGF beta 1, TGF beta 2, and TGF beta 3 mRNAs in MCF-7 human breast cancer cells. Growth of MCF-7 cells was stimulated by norethindrone (10(-8)-10(-5) M), with maximal growth stimulation at 10(-7) M norethindrone after 7 days of treatment. However, the growth of MCF-7 cells was not affected by MPA (10(-8) M) or R5020 (10(-8) M). Treatment with the antiestrogen 4-hydroxytamoxifen at a concentration of 10(-7) M blocked the growth stimulation induced by norethindrone. The norethindrone-induced growth stimulation was accompanied by a dramatic decrease in TGF beta 2 and TGF beta 3 mRNA levels, whereas the level of TGF beta 1 mRNA was not affected by any of the compounds tested. In addition, treatment with MPA or R5020 did not affect TGF beta 2 and TGF beta 3 mRNA levels. The inhibitory effect of norethindrone on TGF beta 2 and TGF beta 3 mRNA levels could be blocked by the addition of 10(-7) M 4-hydroxytamoxifen. Norethindrone as well as estradiol decreased estrogen receptor mRNA levels and increased progesterone receptor mRNA levels. This is the first report which demonstrates that norethindrone stimulates estrogen-responsive human breast cancer cell growth and inhibits the expression of TGF beta 2 and TGF beta 3 mRNAs. These results suggest that the differential regulation of TGF beta expression by norethindrone may be at least partly responsible for the growth stimulation induced by norethindrone. Thus, the norethindrone component of some oral contraceptives may be sufficiently estrogenic to facilitate the development of breast cancer.