Maurizio Gelati

Expression of drug resistance proteins Pgp, MRP1, MRP3, MRP5 AND GST-π in human glioma

SummaryChemotherapy in glioma is poorly effective: the blood–brain barrier and intrinsic and/or acquired drug resistance of tumor cells could partly explain this lack of major effect. We investigated expression of P-glycoprotein (Pgp), multidrug resistance protein (MRP) 1, MRP3, MRP5 and glutathione-S-transferase π (GST-π) in malignant glioma patients. Cytofluorimetric analysis of 48 glioma specimens and 21 primary cultures showed high levels of MRP1, moderate levels of MRP5 and low levels of Pgp, GST-π and MRP3. Immunohistochemistry (25 glioma specimens) showed expression of GST-π (66.7% of cases), MRP1 (51.3%), MRP5 (45.8%), Pgp (34.8%) and MRP3 (29.9%) in tumor cells. Moreover, analysis of tumor samples by real time quantitative PCR showed mRNA expression of all investigated genes. Tumor vasculature, analyzed in glioma specimens and in tumor derived endothelial cells, showed expression of all investigated proteins. Non-tumor brain samples (from a patient with arteriovenous malformation and from one with epilepsy), normal human astrocytes and cultured endothelial cells were also analyzed: astrocytes and endothelial cells expressed the highest levels of the investigated proteins, mainly MRP1 and MRP5. No significant differences in proteins expression were detected between primary or recurrent gliomas, suggesting that glioma chemoresistance is mostly intrinsic. Therefore, we detected, for the first time, the presence of MRP3 and MRP5 on glioma specimens – both in tumor and endothelial cells – and we delineated an expression profile of chemoresistance proteins in glioma. The possible association of inhibitors of drug efflux pumps with chemotherapy could be investigated to improve drugs delivery into the tumor and their cytotoxic effects.

Intracavitary VEGF, bFGF, IL-8, IL-12 levels in primary and recurrent malignant glioma.

Intracavitary levels of VEGF, bFGF, IL-8 and IL-12 were evaluated by ELISA in 45 patients, 7 with recurrent anaplastic astrocytoma (rAA), 12 with glioblastoma (GBM) and 26 with recurrent glioblastoma (rGBM). In 25 patients plasma levels of the molecules were also quantitated. Twenty-three healthy controls were also studied for plasma concentrations of the same molecules.Plasma levels of VEGF (mean 33.89 ± 6.71 pg/ml) and bFGF (mean 11.1 ± 3.24 pg/ml) were higher in patients than in controls (mean 16.78 ± 3.7 pg/ml for VEGF, mean 0.21 ± 0.09 pg/ml for bFGF) (p = 0.04 and p = 0.001, respectively) while plasma IL-12 levels were lower (mean 45.6 ± 1.5 pg/ml in patients, mean 79.7 ± 1.3 pg/ml in controls) (p = 0.009).Intracavitary VEGF levels were 5–53.307 fold higher (mean 90,900 ± 24,789 pg/ml) than in the corresponding plasma. Also IL-8 concentrations were higher in intracavitary fluid (mean 6,349.76 ± 1,460.93 pg/ml) than in plasma (mean 43.44 ± 24.82 pg/ml). Maximum VEGF levels were found in tumor fluid of recurrent glioblastoma patients (mean 147,678 ± 39,903 pg/ml), intermediate levels in glioblastoma patients (mean 20,322 ± 11,892 pg/ml) and lower levels in rAA patients (mean 9,111 ± 5,789 pg/ml). The data also suggest that higher intracavitary levels of VEGF and IL-8, and lower IL-12 levels, may be correlated with shorter adjunctive survival times, but more data will need to be collected to establish this correlation clearly.

CXCL12 in malignant glial tumors: a possible role in angiogenesis and cross-talk between endothelial and tumoral cells

CXCL12 (stromal cell-derived factor-1/CXCL12) regulates leukocyte, endothelial and hematopoietic precursor migration, bone-marrow myelopoiesis and angiogenesis. CXCL12 and its receptor CXCR4 are over-expressed in malignant gliomas, which are highly vascularized tumors with a poor prognosis.We studied the expression of CXCL12 and CXCR4 in glioma cell lines, endothelial cells, tissue sections and endocavitary fluids from patients with gliomas.We then analyzed the proliferative and the apoptotic effect of CXCL12 in endothelial cells and glioma primary cultures.We observed the release of CXCL12 in supernatants of human brain microvascular endothelial cells and at variable levels, in post-surgical endocavitary fluids. CXCL12 was expressed in both glioma and endothelial cells as assessed by immunostaining of surgical brain sections. CXCR4 was found in cells lines and primary cultures from malignant gliomas as well as in endothelial cells and was increased by vascular endothelial growth factor and basic fibroblast growth factor (bFGF). CXCL12 inhibited bFGF-induced proliferation of endothelial cells and increased the survival of endothelial cells. The survival of primary cells obtained from glioma specimens was also enhanced in the presence of CXCL12.We point out the presence and the release of CXCL12 in tumor microenvironment and we observed a modu\-lating effect of CXCL12 on proliferation and survival of both endothelial and tumoral cells. Our data support in vivo studies suggesting a role in angiogenesis played by CXCL12, which could represent a possible prognostic factor.

Expression and Modulation of IFN-γ-Inducible Chemokines (IP-10, Mig, and I-TAC) in Human Brain Endothelium and Astrocytes: Possible Relevance for the Immune Invasion of the Central Nervous System and the Pathogenesis of Multiple Sclerosis

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system (CNS) mediated by blood-derived immune cells invading the CNS. This invasion could be determined by chemokines, and their role within the MS-affected brain is still poorly defined. We investigated the expression by RT-PCR and protein release by ELISA of the interferon-gamma (IFN-gamma)-inducible chemokines in human brain microvascular endothelial cells (HBMECs) and astrocytes. The monokine induced by IFN-gamma (Mig) behaves as a homing chemokine constitutively expressed in HBMECs and astrocytes, whereas the IFN-gamma-inducible 10-kDa protein (IP-10) and IFN-inducible T cell alpha-chemoattractant (I-TAC) are induced only after inflammatory stimuli. The biologic activity of IFN-gamma-inducible chemokines from an endothelial source was analyzed, and the transendothelial migration of activated lymphocytes was partly antagonized by specific antibodies, especially anti-Mig antibody. Our data highlight the capability of cells of the CNS to activate the chemoattractant machinery in a proinflammatory environment and in MS.

Soluble Fas (Apo-1) levels in cerebrospinal fluid of multiple sclerosis patients

CSF and serum levels of soluble Fas were studied in MS patients, in patients with various neurological diseases and in healthy controls. We did not detect differences in serum sFas levels between MS patients and controls. In CSF, despite sFas levels being similar in all patients studied, a statistically significant correlation between sFas CSF/sFas serum ratio and BBB damage (expressed as albumin CSF/albumin serum ratio) was detected in non-MS neurological disease, but not in MS patients. The normalized ratio (sFas CSF/sFas serum)/(Alb CSF/Alb serum) was significantly increased in MS patients compared to patients with non-inflammatory neurological disease suggesting an intrathecal synthesis of soluble Fas in MS. The percentage of apoptotic mononuclear cells was higher in CSF as compared to peripheral blood; moreover a lower proportion of apoptotic cells was found in CSF of MS patients. The findings lend support to the involvement of sFas in MS pathogenesis and suggest that a lower apoptosis in CSF may be a feature of the disease.

Transforming Growth Factor-β1 and CD105 Promote the Migration of Hepatocellular Carcinoma–Derived Endothelium

Hepatocellular carcinoma (HCC) is one of most malignant and aggressive human tumors. Transforming growth factor-beta1 (TGF-beta1) and its coreceptor CD105 have been shown to contribute to HCC malignant progression. TGF-beta1 and CD105 have also been implicated in angiogenesis, but their role in the vascularization of HCC has not been investigated. To fill this gap, we studied the effect of TGF-beta1 and CD105 on HCC-derived endothelium. By using immunomagnetic beads, we isolated and cultured endothelial cells (ECs) from HCC (HCC-EC) and adjacent nonneoplastic tissue (nNL-ECs) obtained from 24 liver biopsies. HCC and nNL biopsies were also analyzed by immunohistochemistry for the expression of CD105, TGF-beta1, Ve-cadherin (Ve-cad), CD44, beta-catenin, and E-cadherin. Compared with nNL-ECs, HCC-ECs had higher expression of CD105, enhanced spontaneous motility, and greater capacity to migrate in response to TGF-beta1 (5 ng/mL), particularly in the presence of a fibronectin matrix. The chemotactic effect of TGF-beta1 was blocked by anti-CD105 antibodies and correlated with the grade of HCC malignancy. Histologic examination of HCC biopsies showed that HCCs with the worse malignant features had the highest expression of TGF-beta1, CD105, and angiogenic markers (Ve-cad and CD44). Because CD105 was highly expressed in microvessels at the tumor periphery and TGF-beta1 staining was only found in neoplastic hepatocytes, we conclude that HCC-derived TGF-beta1 may act as a chemoattractant for CD105-expressing ECs and as a promoter of tumor angiogenesis. Thus, drugs that selectively target the TGF-beta1/CD105 axis may interfere with HCC-related angiogenesis and HCC progression.

Lycopersicon esculentum lectin: an effective and versatile endothelial marker of normal and tumoral blood vessels in the central nervous system

The binding of Lycopersicon esculentum lectin (LEA) to the vascular endothelium was studied in the central nervous system of rat, mouse and guinea pig at different developmental ages, and in a gliosarcoma model. Our observations showed that LEA consistently stained the entire vascular tree in the spinal cord and in the brain of all animal species at all developmental ages investigated. In the tumor model, the staining of the vascular network was very reproducible, enabled an easy identification of vascular profiles and displayed a higher efficiency when compared to two other commonly used vascular marker (EHS laminin and PECAM-1). Moreover, our results showed that LEA staining was comparable in both vibratome and paraffin sections and could be easily combined with other markers in double labeling experiments. These observations indicate that LEA staining may represent an effective and versatile endothelial marker for the study of the vasculature of the central nervous system in different animal species and experimental conditions.

CXCL12 expression is predictive of a shorter time to tumor progression in low-grade glioma: a single-institution study in 50 patients.

SummaryThe clinical course of 50 patients with low-grade glioma (31 male, 19 female) undergoing surgery at a single Institution from 1992 to 1996 was analyzed in relationship with known prognostic factors as far as time to tumor progression (TTP) and survival time (ST) are concerned. Moreover, microvessel density (MVD) and expression of the angiogenesis-related chemokine CXCL12 were investigated in surgical specimens. Age at diagnosis ranged from 1 to 68 years (median 30). Histology revealed 11 fibrillary, 6 protoplasmatic, 5 gemistocytic astrocytoma, 18 oligoastrocytoma and 10 oligodendroglioma. Mean follow-up was 86 months. Four patients were lost to follow-up. Of the remaining 46, twenty-four have shown disease progression and 14 have died. Median overall survival was not achieved; an estimated 75% percentage of survivors was found at 78 months. Complete gross tumor removal was associated to a longer TTP (P = 0.04 logrank). Of the investigated immunohistochemical parameters, while MVD was not predictive of subsequent TTP, expression of CXCL12 was associated with a significantly shorter TTP (P = 0.01 logrank): this predictive value remained significant (P = 0.02) at multivariate analysis. The data suggest the possible prognostic value for CXCL-12 (an angiogenesis- and tumor-growth-related chemokine) on TTP in low-grade gliomas.

Effects of β-IFN-1b treatment in MS patients on adhesion between PBMNCs, HUVECs and MS-HBECs: an in vivo and in vitro study

Abstract The in vivo effects on the expression of adhesion molecules and on the adhesion between mononuclear cells and multiple sclerosis human brain endothelial cells (MS-HBECs) were investigated at the beginning of β-IFN-1b treatment of MS patients. MS-HBECs were isolated from a surgical specimen obtained from an MS patient undergoing brain surgery for vascular aneurysm. 48 h after the first single administration of β-IFN-1b, PBMNCs of 10 MS patients were analyzed for HLA-DR, CD11a, CD18 and VLA-4 expression and the adhesion between PBMNCs and both stimulated and unstimulated MS-HBECs evaluated. sICAM-1 and sVCAM-1 dosage in the serum of the patients was checked as well. The experiments were repeated using HUVECs in order to detect possible endothelial organ-specific differences. The experiments were also performed after six months of β-INF-1b treatment on HUVECs. No significant effects on mononuclear cells/endothelium adhesion were detected at 48 h, but adhesion of PBMNCs to HUVECs decreased at six months. An increase in HLA-DR and VLA-4 and a decrease of CD18 was detected in monocytes. The serum level of sVCAM-1 increased at T2 and was still higher than at T0 at six months. The effect of the β-IFN-1b treatment on both MS-HBECs and HUVECs, was selectively studied in vitro by testing the expression of cytokine-induced adhesion molecules HLA-DR, ICAM-1 and VCAM-1. The in vitro experiments confirmed that β-IFN-1b is able to antagonize γ-IFN-induced HLA-DR expression on MS human brain endothelial cells without relevant effects on VCAM-1 and ICAM-1.

High-dose methylprednisolone reduces cytokine-induced adhesion molecules on human brain endothelium

OBJECTIVE We investigated the in vitro effects of low- and high-dose methylprednisolone (MP) on the cytokine-induced expression of HLA-DR, ICAM-1 and VCAM-1 on human brain microvessel endothelial cells (HBMECs). METHODS Brain endothelium was obtained from microvessels included in the apparently normal white matter of surgical specimens of nine patients. Cells were stained with monoclonal antibodies anti-HLA-DR, anti-ICAM-1 and anti-VCAM-1 and analysed by flow cytometry as fluorescence histograms. The mean fluorescence intensity (MFI) of HBMECs treated with different stimuli was calculated. RESULTS gamma-IFN-induced HLA-DR was down-regulated in a dose-dependent manner by MP. High-dose MP reduced the TNF-alpha-induced ICAM-1 and VCAM-1 expression. CONCLUSIONS The down-regulation of adhesion molecules on cerebral endothelial cells could decrease mononuclear cell transmigration through the blood brain barrier and consequently the perivascular infiltrates. The results add support to the rationale for high-dose MP treatment in multiple sclerosis relapses.