Dipak K. Banerjee

Biphasic estrogen response on bovine adrenal medulla capillary endothelial cell adhesion, proliferation and tube formation

Abnormal angiogenesis underlies many pathological conditions and is critical for the growth and maintenance of various types of tumors, including hormone-dependent cancers. Since estrogens are potent carcinogens in humans and rodents, and are involved in regulating angiogenesis, this study was designed to examine the effect of estrogen on the behavior of an established bovine capillary endothelial cell line, a simple and physiologically relevant model of the capillary wall. The results demonstrate that 17β-estradiol (E2), at different conditions, exerts both stimulatory and inhibitory effects on endothelial cell adhesion, proliferation and tube formation in vitro. Utilizing a cellular attachment assay, chronic exposure to nanomolar concentrations of E2 (i.e. 1 and 10 nM) increased endothelial cell adhesion significantly compared to vehicle treated controls. Cellular adhesion was inhibited by micromolar concentrations of E2. Cell count, PCNA immunohistochemistry and Western blot analysis demonstrated enhanced cell proliferation at low E2 concentration in estrogen-deplete medium. Inhibition of cellular proliferation was observed in both estrogen-replete and deplete medium at higher E2 concentrations (i.e. 1 and 10 µM). Furthermore, in vitro tube formation increased up to 3.0 fold in the presence of 10 nM and higher E2 concentrations. The present observations indicate that in vitro regulation of capillary endothelial cell adhesion, proliferation and capillary tube formation by estrogen, are dose dependent.

Steroid regulation of monoamine oxidase activity in the adrenal medulla.

Administration of different steroid hormones in vivo has distinct and specific effects on the MAO activity of the adrenal medulla. In an effort to reconstitute these effects in defined cells, we have isolated endothelial cells and chromaffin cells from the bovine adrenal medulla and tested each cell type for sensitivity to these steroids. As in the intact animal, we found that endothelial cell MAO activity was stimulated 1.5‐ to 2.5‐fold by 10 μM progesterone, hydrocortisone, and dexamethasone, inhibited by ca. 50% by 17‐α‐estradiol, but unaffected by testosterone. The type of MAO in the endothelial cells was found to be exclusively of the A type. The chromaffin cells had MAO B exclusively and were inert to treatment with dexamethasone. The mode of action of the various steroids on MAO A activity in endothelial cells seemed to be that of affecting the number of MAO molecules, as binding of [3H]pargyline, an MAO inhibitor, changed in proportion to changes in enzyme activity. Consistently, the kinetic parameters for MAO A showed changes in Vmax but not Km under all conditions. The specificity of steroid action on MAO A activity was also supported by the fact that steroid‐induced changes in total cell division ([14C]thy‐midine incorporation) and total protein synthesis ([14C]leucine incorporation) were seen after changes in MAO A. We conclude that the differential effects of steroids on MAO activity in the intact adrenal medulla can be reproduced in cultured adrenal medullary endothelial cells but not in chromaffin cells. Therefore we suggest that the action of these steroid hormones on the intact adrenal medulla may be restricted to the endothelial cell component of this tissue.—Youdim, M. B. H.; Banerjee, D. K.; Kelner, K.; Offutt, L.; Pollard, H. B. Steroid regulation of MAO activity in the adrenal medulla. FASEB J. 3: 1753‐1759; 1989.

Unfolded protein response is required in nu/nu mice microvasculature for treating breast tumor with tunicamycin.

Up-regulation of the dolichol pathway, a “hallmark” of asparagine-linked protein glycosylation, enhances angiogenesis in vitro. The dynamic relationship between these two processes is now evaluated with tunicamycin. Capillary endothelial cells treated with tunicamycin were growth inhibited and could not be reversed with exogenous VEGF165. Inhibition of angiogenesis is supported by down-regulation of (i) phosphorylated VEGFR1 and VEGFR2 receptors; (ii) VEGF165-specific phosphotyrosine kinase activity; and (iii) MatrigelTM invasion and chemotaxis. In vivo, tunicamycin prevented the vessel development in MatrigelTM implants in athymic Balb/c (nu/nu) mice. Immunohistochemical analysis of CD34 (p < 0.001) and CD144 (p < 0.001) exhibited reduced vascularization. A 3.8-fold increased expression of TSP-1, an endogenous angiogenesis inhibitor in MatrigelTM implants correlated with that in tunicamycin (32 h)-treated capillary endothelial cells. Intravenous injection of tunicamycin (0.5 mg/kg to 1.0 mg/kg) per week slowed down a double negative (MDA-MB-435) grade III breast adenocarcinoma growth by ∼50–60% in 3 weeks. Histopathological analysis of the paraffin sections indicated significant reduction in vessel size, the microvascular density and tumor mitotic index. Ki-67 and VEGF expression in tumor tissue were also reduced. A significant reduction of N-glycan expression in tumor microvessel was also observed. High expression of GRP-78 in CD144-positive cells supported unfolded protein response-mediated ER stress in tumor microvasculature. ∼65% reduction of a triple negative (MDA-MB-231) breast tumor xenograft in 1 week with tunicamycin (0.25 mg/kg) given orally and the absence of systemic and/or organ failure strongly supported tunicamycins potential for a powerful glycotherapeutic treatment of breast cancer in the clinic.

Potentiation of angiogenic switch in capillary endothelial cells by cAMP : A cross-talk between up-regulated LLO biosynthesis and the HSP-70 expression

During tumor growth and invasion, the endothelial cells from a relatively quiescent endothelium start proliferating. The exact mechanism of switching to a new angiogenic phenotype is currently unknown. We have examined the role of intracellular cAMP in this process. When a non-transformed capillary endothelial cell line was treated with 2 mM 8Br-cAMP, cell proliferation was enhanced by ∼70%. Cellular morphology indicated enhanced mitosis after 32–40 h with almost one-half of the cell population in the S phase. Bcl-2 expression and caspase-3, -8, and -9 activity remained unaffected. A significant increase in the Glc3Man9GlcNAc2-PP-Dol biosynthesis and turnover, Factor VIIIC N-glycosylation, and cell surface expression of N-glycans was observed in cells treated with 8Br-cAMP. Dol-P-Man synthase activity in the endoplasmic reticulum membranes also increased. A 1.4–1.6-fold increase in HSP-70 and HSP-90 expression was also observed in 8Br-cAMP treated cells. On the other hand, the expression of GRP-78/Bip was 2.3-fold higher compared to that of GRP-94 in control cells, but after 8Br-cAMP treatment for 32 h, it was reduced by 3-fold. GRP-78/Bip expression in untreated cells was 1.2–1.5-fold higher when compared with HSP-70 and HSP-90, whereas that of the GRP-94 was 1.5–1.8-fold lower. After 8Br-cAMP treatment, GRP-78/Bip expression was reduced 4.5–4.8-fold, but the GRP-94 was reduced by 1.5–1.6-fold only. Upon comparison, a 2.9-fold down-regulation of GRP-78/Bip was observed compared to GRP-94. We, therefore, conclude that a high level of Glc3Man9GlcNAc2-PP-Dol, resulting from 8Br-cAMP stimulation up-regulated HSP-70 expression and down-regulated that of the GRP-78/Bip, maintained adequate protein folding, and reduced endoplasmic reticulum stress. As a result capillary endothelial cell proliferation was induced.

Low expression of lipid-linked oligosaccharide due to a functionally altered Dol-P-man synthase reduces protein glycosylation in cAMP-dependent protein kinase deficient Chinese hamster ovary cells

Chinese hamster ovary cells express a wide variety of glycoproteins with Mr ranging from 15,000 to 200,000 dalton and higher. Glycosylation of these proteins was much less in cAMP-dependent protein kinase (PKA)-deficient mutants which expressed either (i) a defective C-subunit with altered substrate specificity and having no detectable type II kinase (mutant 10215); or (ii) an altered RI subunit and having no detectable type II kinase (mutant 10248); or (iii) exhibited the lowest level of total kinase with no detectable type I kinase but having a small amount of type II kinase (mutant 10260). Addition of 8Br-cAMP enhanced protein glycosylation index in wild type cells 10001 by 120% but only 7 to 23% in the mutant cells. The rate of lipid-linked oligosaccharide (LLO) biosynthesis was linear for 1 h in all cell types, but the total amount of LLO expressed was much less in PKA-deficient mutants. Pulse-chase experiments indicated that the t1/2 for LLO turnover was also twice as high in PKA-deficient cells as in the wild type. Size exclusion chromatography of the mild-acid released oligosaccharide confirmed that both wild type and the mutant cells synthesized Glc3Man9GlcNAc2-PP-Dol as the most predominating species with no accumulation of Man5GlcNAc2-PP-Dol in the mutants. Kinetic studies exhibited a reduced mannosylphosphodolichol synthase (DPMS) activity in mutant cells with a Km for GDP-mannose 160 to 400% higher than that of the wild type. In addition, the kcat for DPMS was also reduced 2 to 4-fold in these mutant cells. Exogenously added Dol-P failed to rescue the kcat for DPMS in CHO cell mutants; however, in vitro protein phosphorylation with a cAMP-dependent protein kinase restored their kinetic activity to the level of the wild type. Published in 2004.

Expression of blood clotting Factor VIII:C gene in capillary endothelial cells

The essential role of Factor VIII:C (FVIII:C, anti‐hemophilia factor A) as a cofactor for Factor IXa‐dependent activation of Factor X has been established. In this paper, we describe that capillary endothelial cells from bovine adrenal medulla express active FVIII:C gene. Accumulation of FVIII:C in conditioned media from an 8‐day‐old culture is approximately twice as high as that stored in the cell when immunoprecipitated FVIII:C was analyzed for its ability to convert Factor X to Factor Xa. Analysis of [35S]methionine‐labeled and immunoprecipitated FVIII:C from cells or conditioned media on SDS‐PAGE under fully denatured conditions indicated that the newly synthesized FVIII:C consists of heavy chain of M r 200,000 and light chain of M r 46,000. The secreted FVIII:C in the non‐reduced condition however, has a molecular weight of 270,000 which suggests that in native protein, the heavy and light chains are held together by S‐S bonds. Furthermore, susceptibility of the immunoprecipitated FVIII:C to N‐glycanase digestion establishes that the endothelial cells derived FVIII:C contains asparagine‐linked carbohydrate side chains.

Beta-adrenoreceptors of multiple affinities in a clonal capillary endothelial cell line and its functional implication.

Abstractβ-Adrenoreceptor has been studied in a clonal capillary endothelial cell line established from the vascular bed of the bovine adrenal medulla. [3H]Dihydroalprenolol ([3H]DHA) binding to the isolated plasma membranes from these cells has demonstrated the presence of β-adrenoreceptors with two different affinities. the dissociation constants (Kd) have been found to be 0.27±0.09×10−9 M and 2.96±0.31×10−9 M, respectively with the corresponding Bmax of 5.1±0.05 and 70.0±0.2 pmol/mg protein, respectively. Inhibition of [3H]DHA binding to the β-receptor by atenolol (a β1-antagonist) and ICI 118,551 (a β1-antagonist) has suggested that the IC50cor (=Ki) for atenolol and ICI 118,551 for high affinity site are 0.08±0.03×10−12 M and 0.25±0.08×10−12 M, respectively. This, therefore, indicates that both atenolol and ICI 118,551 are able to displace the bound ligand effectively but the β1-selective antagonist atenolol is 3 times more potent than its β2 counterpart, ICI 118,551. Displacement of [3H]DHA binding to the endothelial cell plasma membrane by the agonists isoproterenol, epinephrine and norephinephrine has established a relative order of Ki for these agents as isoproterenol (0.56±0.19×10−9 M)-norepinephrine (0.71±0.24×10−9 M) for the high affinity site. The corresponding values for the low affinity site, however, are 4.62±0.64×10−9 M, 6.21±0.86×10−9 M and 5.90±0.82×10−9 M, respectively for the same agonists. Increased intracellular cAMP accompanied with cellular proliferation in the presence of isoproterenol has suggested not only the coupling of β-adrenoreceptors to the adenylate cyclase system but also its involvement in endothelial cell proliferation.

Mannosylphosphodolichol Synthase Activity is Associated with a 32 kDa Phosphoprotein

Failure of actinomycin D to block the activation of Dol-P-Man synthase in isoproterenol-treated capillary endothelial cells, supported that isoproterenol effect was not mediated by active transcription of the Dol-P-Man synthase gene during a short-term β-adrenoreceptor stimulation. Instead, it was a net effect of protein phosphorylation by cAMP-dependent protein kinase. Using antibody as a probe we have now demonstrated that Dol-P-Man synthase activity is associated with a 32 kDa ER phosphoprotein.

Mannosylphosphodolichol synthase overexpression supports angiogenesis

Abstract Mannosylphosphodolichol synthase (DPMS) plays a critical role in Glc3Man9GlcNAc2-PP-Dol (lipid-linked oligosaccha-ride, LLO) biosynthesis, an essential intermediate in asparagine-linked (N-linked) protein glycosylation. We observed earlier that phosphorylation of DPMS increases the catalytic activity of the enzyme by increasing the Vmax as well as the enzyme turnover (kcat) without significantly changing the Km for GDP-mannose. As a result, LLO biosynthesis, turnover and protein N-glycosylation are increased. This is manifested in increased proliferation of capillary endothelial cells, i.e. angiogenesis. We have then asked, if the phosphorylation event or the upregulation of DPMS due to overproduction of the enzyme is the key factor in upregulating angiogenesis? This question has been answered by isolating a stable capillary endothelial cell clone overexpressing the gene encoding DPMS. Our results indicate that the DPMS-overexpressing clone has a high level of DPMS mRNA as judged by QRT-PCR. The clone also expresses nearly four times more DPMS protein than the clone transfected with pEGFP-N1 vector only (i.e. control) as analyzed by Western blotting. Most importantly, the overexpressing DPMS clone has ∼108% higher DPMS activity than the vector control. Immunofluorescence microscopy with Texas Red-conjugated wheat germ agglutinin indicates a high level of expression of (GlcNAc-β-(1,4)-GlcNAc) 1-4-β-GlcNAc-NeuAc glycans on the external surface of the capillary endothelial cells overexpressing DPMS. Increased cellular proliferation and accelerated healing of the wound induced by mechanical stress of the DPMS-overexpressing clone unequivocally supports a role of DPMS in angiogenesis.

Insulin up-regulates a Glc3Man9GlcNAc2-PP-Dol pool in capillary endothelial cells not essential for angiogenesis

Endothelial cells line blood vessels, and their proliferation during neovascularization (i.e., angiogenesis) is essential for a normal growth and development as well as for tumor progression and metastasis. Mechanistic details indicated that down-regulation of Glc3Man9GlcNAc2-PP-Dol level reduced angiogenesis and induced apoptosis in capillary endothelial cells (Martínez JA, Torres-Negrón I, Amigó LA, Banerjee DK, Cellular and Molec Biochem 45, 137–152 (1999)). Unlike in any other insulin-responsive cells, insulin reduced capillary endothelial cell proliferation by increasing the cell doubling time. But, when analyzed, the rate of lipid-linked oligosaccharide-PP-Dol (LLO) synthesis as well as its turnover (i.e., t1/2) were increased in insulin treated cells. No major differences in their molecular size were observed. This corroborated with an enhanced glycosylation of Factor VIIIC, an N-linked glycoprotein (essential cofactor of the blood coagulation cascade) and a marker for the capillary endothelial cell. Increased LLO synthesis was independent of elevating either Dol-P level or Man-P-Dol synthase gene (dpm) transcription. Insulin however, enhanced 2-deoxy-glucose transport across the endothelial cell plasma membrane and caused increased secretion of Factor VIIIC, thus, supporting the existence of additional LLO pool(s), and arguing favorably that growth retardation of capillary endothelial cells by insulin turned a highly proliferative cell into a highly secretory cell. Published in 2004.