Federica Barchiesi

In vivo antigen loading and activation of dendritic cells via a liposomal peptide vaccine mediates protective antiviral and anti-tumour immunity

Initiation of antiviral and anti-tumour T cell responses is probably achieved mainly by dendritic cells (DC) transporting antigen from the periphery into organised lymphoid tissues. To develop T cell vaccines it is, therefore, important to understand the accessibility of the antigen to DC in vivo and whether DC are activated by vaccination. Here we have evaluated the immunogenicity of a liposomal vaccine formulation with antigenic peptides derived from the glycoprotein of the lymphocytic choriomeningitis virus. Liposome-encapsulated peptides were highly immunogenic when administered intradermally and elicited protective antiviral immunity. After intradermal injection, liposomes formed antigen depots which facilitated long-lasting in vivo antigen loading of dendritic cells almost exclusively in the local draining lymph nodes. The immunogenicity of the liposomal peptide vaccine was further enhanced by incorporation of immunostimulatory oligonucleotides leading to activation of DC. This optimised liposomal peptide vaccine elicited also anti-tumour immunity and induced CTL responses comparable to adoptively transferred, peptide-presenting DC. Thus, our data show that liposomal formulations of peptide vaccines are highly effective at direct in vivo antigen loading and activation of DC leading to protective antiviral and anti-tumour immune responses.

2-Methoxyestradiol, an Estradiol Metabolite, Inhibits Neointima Formation and Smooth Muscle Cell Growth via Double Blockade of the Cell Cycle

2-Methoxyestradiol (2-ME), an endogenous metabolite of estradiol with no affinity for estrogen receptors, is a potent anticarcinogenic agent (in phase II clinical trials) and mediates the inhibitory effects of estradiol on smooth muscle cell (SMC) growth. Here we studied the intracellular mechanisms by which 2-ME inhibits SMC growth and whether 2-ME prevents injury-induced neointima formation. 2-ME concentrations that inhibit proliferation of cycling human aortic SMCs by ≥50% blocked cell-cycle progression in G0/G1 and in G2/M phase, as determined by flow cytometry. Consistent with the cell-cycle effects, at a molecular level (Western blots), 2-ME inhibited cyclin D1 and cyclin B1 expression; cyclin-dependent kinase (cdk)-1 and cdk-2 activity; and retinoblastoma protein (pRb), extracellular signal-regulated kinase (ERK) 1/2, and Akt phosphorylation. 2-ME also upregulated the Cdk inhibitor p27 and interfered with tubulin polymerization. Moreover, 2-ME augmented COX-2 expression, suggesting that it may also inhibit SMC growth via prostaglandin formation. In rats, treatment with 2-ME abrogated injury-induced neointima formation; decreased proliferating SMCs; downregulated expression of proliferating-cell nuclear antigen (PCNA), c-myc, cyclin D1, cyclin B1, phosphorylated Akt, phosphorylated ERK1/2, p21, and pRb; inhibited cdk-1 and cdk-4 activity; and upregulated expression of cyclooxygenase (COX)-2 and p27. Caspase-3 cleavage assay and fluorescence-activated cell-sorting (FACS) analysis showed no evidence of apoptosis in 2-ME–treated SMCs, and TUNEL staining in carotid segments showed no evidence of 2-ME–induced apoptosis in vivo. The antimitotic effects of 2-ME on SMCs are mediated by the inhibition of key cell-cycle regulatory proteins and effects on tubulin polymerization and COX-2 upregulation. These effects of 2-ME most likely contribute to the antivasoocclusive actions of this endogenous compound.

Methoxyestradiols Mediate Estradiol-Induced Antimitogenesis in Human Aortic SMCs

Estrogen receptors (ERs) are considered to mediate the ability of 17&bgr;-estradiol (estradiol) to reduce injury-induced proliferation of vascular smooth muscle cells (VSMCs), leading to vascular lesions. However, the finding that estradiol attenuates formation of vascular lesions in response to vascular injury in knockout mice that lack either ER-&agr; or ER-&bgr; challenges this concept. Our hypothesis is that the local metabolism of estradiol to methoxyestradiols, metabolites of estradiol with little affinity for ERs, mediates the ER-independent antimitogenic effects of estradiol on VSMCs. In human VSMCs, 2-methoxyestradiol and 2-hydroxyestradiol were more potent than was estradiol in inhibiting DNA synthesis (3[H]-thymidine incorporation), collagen synthesis (3[H]-proline incorporation), cell proliferation (cell number), and cell migration (movement of cells across a polycarbonate membrane). The inhibitory effects of estradiol on VSMCs were enhanced by cytochrome-P450 (CYP450) inducers 3-methylcholanthrene and phenobarbital. Moreover, the inhibitory effects of estradiol were blocked in the presence of the CYP450 inhibitor 1-aminobenzotriazole and the catechol-O-methyltransferase inhibitors quercetin and OR486. Both OR486 and quercetin blocked the conversion of 2-hydroxyestradiol to 2-methoxyestradiol; moreover, they blocked the antimitogenic effects of 2-hydroxyestradiol but not of 2-methoxyestradiol. The ER antagonist ICI182780 blocked the inhibitor effects of estradiol on VSMCs, but only at concentrations (>50 &mgr;mol/L) that also inhibit the metabolism of estradiol to hydroxyestradiols (precursors of methoxyestradiols). In conclusion, the inhibitory effects of locally applied estradiol on human VSMCs are mediated via a novel ER-independent mechanism involving estradiol metabolism. These findings imply that vascular estradiol metabolism may be an important determinant of the cardiovascular protective effects of estradiol and that nonfeminizing estradiol metabolites may confer cardiovascular protection regardless of gender.

CYP450- and COMT-Derived Estradiol Metabolites Inhibit Activity of Human Coronary Artery SMCs

Abstract—The purpose of this study is to test the hypothesis that the inhibitory effects of estradiol in human coronary vascular smooth muscle cells are mediated via local conversion to methoxyestradiols via specific cytochrome P450s (CYP450s) and catechol-O-methyltransferase (COMT). The inhibitory effects of estradiol on serum-induced cell activity (DNA synthesis, cell number, collagen synthesis, and cell migration) were enhanced by 3-methylcholantherene, phenobarbital (broad-spectrum CYP450 inducers), and &bgr;-naphthoflavone (CYP1A1/1A2 inducer) and were blocked by 1-aminobenzotriazole (broad-spectrum CYP450 inhibitor). Ellipticine, &agr;-naphthoflavone (selective CYP1A1 inhibitors), and pyrene (selective CYP1B1 inhibitor), but not ketoconazole (selective CYP3A4 inhibitor) or furafylline (selective CYP1A2 inhibitor), abrogated the inhibitor effects of estradiol on cell activity, a profile consistent with a CYP1A1/CYP1B1-mediated mechanism. The inhibitory effects of estradiol were blocked by the COMT inhibitors OR486 and quercetin. The estrogen receptor antagonist ICI 182,780 blocked the inhibitory effects of estradiol, but only at concentrations that also blocked the metabolism of estradiol to hydroxyestradiols (precursors of methoxyestradiols). Western blot analysis revealed that coronary smooth muscle cells expressed CYP1A1 and CYP1B1. Moreover, these cells metabolized estradiol to hydroxyestradiols and methoxyestradiols, and the conversion of 2-hydroxyestradiol to 2-methoxyestradiol was blocked by OR486 and quercetin. These findings provide evidence that the inhibitory effects of estradiol on coronary smooth muscle cells are largely mediated via CYP1A1- and CYP1B1-derived hydroxyestradiols that are converted to methoxyestradiols by COMT.

Methoxyestradiols Mediate the Antimitogenic Effects of 17β-Estradiol Direct Evidence From Catechol-O-Methyltransferase–Knockout Mice

Background—Studies using pharmacological agents suggest but do not prove that the antimitogenic effects of estradiol are caused by conversion of estradiol to hydroxyestradiols (mediated by CYP450s) followed by methylation of hydroxyestradiols to methoxyestradiols (mediated by catechol-O-methyltransferase, COMT). Methods and Results—To test this hypothesis more rigorously, we used aortic smooth muscle cells (SMCs) from mice lacking COMT (COMT-KO). Wild-type (WT) but not COMT-KO SMCs efficiently converted 2-hydroxyestradiol to 2-methoxyestradiol. Both WT and COMT-KO SMCs expressed estrogen receptors. Estradiol and 2-hydroxyestradiol concentration-dependently inhibited serum-induced DNA synthesis, cell numbers, and collagen synthesis in WT but not COMT-KO SMCs. 2-Methoxyestradiol inhibited DNA synthesis, cell numbers, and collagen synthesis in both WT and COMT-KO SMCs. Conclusions—These data provide strong evidence that the vascular antimitogenic effects of estradiol are estrogen receptor–independent and involve the sequential conversion of estradiol to hydroxyestradiols and then to methoxyestradiols.

Candidate Genes and Mechanisms for 2-Methoxyestradiol–Mediated Vasoprotection

2-Methoxyestradiol (2-ME; estradiol metabolite) inhibits vascular smooth muscle cell (VSMC) growth and protects against atherosclerosis and vascular injury; however, the mechanisms by which 2-ME induces these actions remain obscure. To assess the impact of 2-ME on biochemical pathways regulating VSMC biology, we used high-density oligonucleotide microarrays to identify differentially expressed genes in cultured human female aortic VSMCs treated with 2-ME acutely (4 hours) or long term (30 hours). Both single gene analysis and Gene Set Enrichment Analysis revealed 2-ME–induced downregulation of genes involved in mitotic spindle assembly and function in VSMCs. Also, Gene Set Enrichment Analysis identified effects of 2-ME on genes regulating cell-cycle progression, cell migration/adhesion, vasorelaxation, inflammation, and cholesterol metabolism. Transcriptional changes were associated with changes in protein expression, including inhibition of cyclin D1, cyclin B1, cyclin-dependent kinase 6, cyclin-dependent kinase 4, tubulin polymerization, cholesterol and steroid synthesis, and upregulation of cyclooxygenase 2 and matrix metalloproteinase 1. Microarray data suggested that 2-ME may activate peroxisome proliferator-activated receptors (PPARs) in VSMCs, and 2-ME has structural similarities with rosiglitazone (PPAR&ggr; agonist). However, our finding of weak activation and lack of binding of 2-ME to PPARs suggests that 2-ME may modulate PPAR-associated genes via indirect mechanisms, potentially involving cyclooxygenase 2. Indeed, the antimitogenic effects of 2-ME at concentrations that do not inhibit tubulin polymerization were blocked by the PPAR antagonist GW9662 and the cyclooxygenase 2 inhibitor NS398. Finally, we demonstrated that 2-ME inhibited hypoxia-inducible factor 1&agr;. Identification of candidate genes that are positively or negatively regulated by 2-ME provides important leads to investigate and better understand the mechanisms by which 2-ME induces its vasoprotective actions.

Estradiol Stimulates Capillary Formation by Human Endothelial Progenitor Cells: Role of Estrogen Receptor-α/β, Heme Oxygenase 1, and Tyrosine Kinase

Endothelial progenitor cells (EPCs) repair damaged endothelium and promote capillary formation, processes involving receptor tyrosine kinases (RTKs) and heme oxygenase 1 (HO-1). Because estradiol augments vascular repair, we hypothesize that estradiol increases EPC proliferation and capillary formation via RTK activation and induction of HO-1. Physiological concentrations of estradiol (10 nmol/L) increased EPC-induced capillary sprout and lumen formation in matrigel/fibrin/collagen systems. Propyl-pyrazole-triol (PPT; 100 nmol/L; estrogen receptor [ER]-&agr; agonist), but not diarylpropionitrile (ER-&bgr; agonist), mimicked the stimulatory effects of estradiol on capillary formation, and methyl-piperidino-pyrazole (ER-&agr; antagonist) abolished the effects of estradiol and PPT. Three different RTK activators (vascular endothelial growth factor, hepatocyte growth factor, and stromal derived growth factor 1) mimicked the capillary-stimulating effects of estradiol and PPT. SU5416 (RTK inhibitor) blocked the stimulatory effects of estradiol and PPT on capillary formation. Estradiol increased HO-1 expression by 2- to 3-fold, an effect blocked by SU5416, and PPT mimicked the effects of estradiol on HO-1. The ability of estradiol to enhance capillary formation, increase expression of HO-1, and augment phosphorylation of extracellular signal–regulated kinase 1/2, Akt, and vascular endothelial growth factor receptor 2 was mimicked by its cell-impermeable analog BSA estradiol. Actinomycin (transcription inhibitor) did not alter the effects of estradiol on RTK activity or vascular endothelial growth factor secretion. We conclude that estradiol via ER-&agr; promotes EPC-mediated capillary formation by a mechanism that involves nongenomic activation of RTKs and HO-1 activation. Estradiol in particular and ER-&agr; agonists in general may promote healing of injured vascular beds by promoting EPC activity leading to more rapid endothelial recovery and capillary formation after injury.

Receptor for Activated Protein Kinase C1 Regulates Cell Proliferation by Modulating Calcium Signaling

Receptor for activated protein kinase C1 (RACK1) is an intracellular scaffolding protein known to interact with the inositol-1,4,5-trisphosphate receptor and thereby enhance calcium release from the sarcoplasmic reticulum. Because calcium signaling may affect vascular smooth muscle cell proliferation, we investigated whether RACK1 regulates proliferation of rat preglomerular microvascular smooth muscle cells. Western blot analysis indicated that preglomerular microvascular smooth muscle cells robustly express RACK1 protein, and coimmunoprecipitation experiments demonstrated that RACK1 binds the inositol-1,4,5-trisphosphate receptor. RACK1 small interfering RNA (siRNA) decreased RACK1 mRNA and protein expression, significantly (P=0.0225) reduced steady-state basal levels of intracellular calcium (6712±156 versus 7408±248, arbitrary fluorescence units in RACK1 siRNA-treated versus control cells, respectively) and significantly (P<0.0001) decreased cell proliferation by ≈50%. Xestospongin C and 2-aminoethoxydiphenyl borate (antagonists of inositol-1,4,5-trisphosphate receptors), cyclopiazonic acid (sarcoplasmic reticulum Ca2+-ATPase inhibitor), and calmidazolium (calmodulin inhibitor) mimicked the effects of RACK1 siRNA on proliferation, and RACK1 siRNA had no additional effects on proliferation in the presence of these agents. RACK1 siRNA did not affect the expression of cyclin D1/2 or phosphorylation of retinoblastoma protein (progrowth cell cycle regulators), yet it caused compensatory decreases in the expression of p21Cip1/Waf1 and p27Kip1 (antigrowth cell cycle regulators). Like preglomerular microvascular smooth muscle cells, glomerular mesangial cells also expressed high levels of RACK1, and RACK1 siRNA inhibited their proliferation. In conclusion, RACK1 modulates proliferation of preglomerular microvascular smooth muscle cells and glomerular mesangial cells, likely via the inositol-1,4,5-trisphosphate receptor/calcium/calmodulin pathway. RACK1 may represent a novel druggable target for treating renal diseases, such as glomerulosclerosis.

Estradiol stimulates capillary formation by human endothelial progenitor cells: role of estrogen receptor-{alpha}/{beta}, heme oxygenase 1, and tyrosine kinase.

Endothelial progenitor cells (EPCs) repair damaged endothelium and promote capillary formation, processes involving receptor tyrosine kinases (RTKs) and heme oxygenase 1 (HO-1). Because estradiol augments vascular repair, we hypothesize that estradiol increases EPC proliferation and capillary formation via RTK activation and induction of HO-1. Physiological concentrations of estradiol (10 nmol/L) increased EPC-induced capillary sprout and lumen formation in matrigel/fibrin/collagen systems. Propyl-pyrazole-triol (PPT; 100 nmol/L; estrogen receptor [ER]-&agr; agonist), but not diarylpropionitrile (ER-&bgr; agonist), mimicked the stimulatory effects of estradiol on capillary formation, and methyl-piperidino-pyrazole (ER-&agr; antagonist) abolished the effects of estradiol and PPT. Three different RTK activators (vascular endothelial growth factor, hepatocyte growth factor, and stromal derived growth factor 1) mimicked the capillary-stimulating effects of estradiol and PPT. SU5416 (RTK inhibitor) blocked the stimulatory effects of estradiol and PPT on capillary formation. Estradiol increased HO-1 expression by 2- to 3-fold, an effect blocked by SU5416, and PPT mimicked the effects of estradiol on HO-1. The ability of estradiol to enhance capillary formation, increase expression of HO-1, and augment phosphorylation of extracellular signal–regulated kinase 1/2, Akt, and vascular endothelial growth factor receptor 2 was mimicked by its cell-impermeable analog BSA estradiol. Actinomycin (transcription inhibitor) did not alter the effects of estradiol on RTK activity or vascular endothelial growth factor secretion. We conclude that estradiol via ER-&agr; promotes EPC-mediated capillary formation by a mechanism that involves nongenomic activation of RTKs and HO-1 activation. Estradiol in particular and ER-&agr; agonists in general may promote healing of injured vascular beds by promoting EPC activity leading to more rapid endothelial recovery and capillary formation after injury.

Estradiol Stimulates Capillary Formation by Human Endothelial Progenitor Cells

Endothelial progenitor cells (EPCs) repair damaged endothelium and promote capillary formation, processes involving receptor tyrosine kinases (RTKs) and heme oxygenase 1 (HO-1). Because estradiol augments vascular repair, we hypothesize that estradiol increases EPC proliferation and capillary formation via RTK activation and induction of HO-1. Physiological concentrations of estradiol (10 nmol/L) increased EPC-induced capillary sprout and lumen formation in matrigel/fibrin/collagen systems. Propyl-pyrazole-triol (PPT; 100 nmol/L; estrogen receptor [ER]-&agr; agonist), but not diarylpropionitrile (ER-&bgr; agonist), mimicked the stimulatory effects of estradiol on capillary formation, and methyl-piperidino-pyrazole (ER-&agr; antagonist) abolished the effects of estradiol and PPT. Three different RTK activators (vascular endothelial growth factor, hepatocyte growth factor, and stromal derived growth factor 1) mimicked the capillary-stimulating effects of estradiol and PPT. SU5416 (RTK inhibitor) blocked the stimulatory effects of estradiol and PPT on capillary formation. Estradiol increased HO-1 expression by 2- to 3-fold, an effect blocked by SU5416, and PPT mimicked the effects of estradiol on HO-1. The ability of estradiol to enhance capillary formation, increase expression of HO-1, and augment phosphorylation of extracellular signal–regulated kinase 1/2, Akt, and vascular endothelial growth factor receptor 2 was mimicked by its cell-impermeable analog BSA estradiol. Actinomycin (transcription inhibitor) did not alter the effects of estradiol on RTK activity or vascular endothelial growth factor secretion. We conclude that estradiol via ER-&agr; promotes EPC-mediated capillary formation by a mechanism that involves nongenomic activation of RTKs and HO-1 activation. Estradiol in particular and ER-&agr; agonists in general may promote healing of injured vascular beds by promoting EPC activity leading to more rapid endothelial recovery and capillary formation after injury.