-
Stefania Elena Navone,
Giovanni Marfia,
Sara Nava,
Gloria Invernici,
Silvia Cristini,
Sergio Balbi,
Simone Sangiorgi,
Emilio Ciusani,
Alessandra Bosutti, Giulio Alessandri,
Mark Slevin,
Eugenio Agostino Parati
[show abstract]
[hide abstract]
ABSTRACT: BACKGROUND: Brain microvascular endothelial cells (BMVECs) constitute the primary limitation for passage of ions and molecules from the blood into the brain through the blood brain barrier. Numerous multi-step procedures for isolating and culturing BMVECs have been described. However, each one demonstrates major limitations in purity of culture and/or low proliferation rate. Our goal was to study the efficiency of our pending patent medium, Endothelial Proliferation Medium (EndoPM), on the isolation and purification of human and murine BMVECs. METHODS: BMVECs, cultured in EndoPM were compared to those cultured in a commercial medium EBM. Cultures were characterized by flow cytometric analysis, lineage differentiation, the ability to form tube-like structure, immunofluorescence, molecular analyses and also in an in vivo model assay. Moreover permeability was assayed by monitoring the passage of Dextran-FITC through a tight monolayer of BMVECs grown to confluence in Boyden chambers. One way Anova two-tailed test was utilized for all statistical analyses. RESULTS: The properties of ECs in human and murine BMVECs is confirmed by the expression of endothelial markers (CD31, CD105, CD146, Tie-2 and vWF), of representative proangiogenic genes (ICAM1, VCAM1 and integrin ITGAV), of considerable tube-forming ability, with low-density lipoprotein uptake, eNOS and GLUT-1 expression. Furthermore cells are able to express markers of the junctional architecture as VE-cadherin, beta-catenin and Claudin-5 and greatly reduce dextran permeability as barrier functional test. Moreover BMVECs spontaneously organize in vascular-like structures and maintain the expression of endothelial markers in an in vivo xenograft model assay. The significant effect of EndoPM is confirmed by the study of proliferation index, survival index and the behaviour of BMVECs and fibroblasts in co-culture conditions. CONCLUSION: Herein we describe a simple and reproducible method for the isolation and expansion of human and mouse BMVECs, based on a newly formulated medium (EndoPM) with optimized concentration of growth factors (EGF, FGF-2 and Bovine Brain Extract-BBE). This procedure should facilitate the isolation and expansion of human and mouse BMVECs with extended lifetime, good viability and purity. This approach may provide an effective strategy to aid phenotypical and functional studies of brain vessels under physiological and pathological conditions.
Vascular cell. 05/2013; 5(1):10.
-
Augusto Pessina,
Valentina Coccè,
Luisa Pascucci,
Arianna Bonomi,
Loredana Cavicchini,
Francesca Sisto,
Maura Ferrari,
Emilio Ciusani,
Antonio Crovace,
Maria Laura Falchetti,
Sonia Zicari,
Arnaldo Caruso,
Stefania Navone,
Giovanni Marfia,
Anna Benetti,
Piero Ceccarelli,
Eugenio Parati, Giulio Alessandri
[show abstract]
[hide abstract]
ABSTRACT: Current leukaemia therapy focuses on increasing chemotherapy efficacy. Mesenchymal stromal cells (MSCs) have been proposed for carrying and delivery drugs to improve killing of cancer cells. We have shown that MSCs loaded with Paclitaxel (PTX) acquire a potent anti-tumour activity. We investigated the effect of human MSCs (hMSCs) and mouse SR4987 loaded with PTX (hMSCsPTX and SR4987PTX) on MOLT-4 and L1210, two leukaemia cell (LCs) lines of human and mouse origin, respectively. SR4987PTX and hMSCsPTX showed strong anti-LC activity. hMSCsPTX, co-injected with MOLT-4 cells or intra-tumour injected into established subcutaneous MOLT-4 nodules, strongly inhibited growth and angiogenesis. In BDF1-mice-bearing L1210, the intraperitoneal administration of SR4987PTX doubled mouse survival time. In vitro, both hMSCs and hMSCsPTX released chemotactic factors, bound and formed rosettes with LCs. In ultrastructural analysis of rosettes, hMSCsPTX showed no morphological alterations while the attached LCs were apoptotic and necrotic. hMSCs and hMSCsPTX released molecules that reduced LC adhesion to microvascular endothelium (hMECs) and down-modulated ICAM1 and VCAM1 on hMECs. Priming hMSCs with PTX is a simple procedure that does not require any genetic cell manipulation. Once the effectiveness of hMSCsPTX on established cancers in mice is proven, this procedure could be proposed for leukaemia therapy in humans.
British Journal of Haematology 01/2013; · 4.94 Impact Factor
-
Francesca Caccuri,
Cinzia Giagulli,
Antonella Bugatti,
Anna Benetti, Giulio Alessandri,
Domenico Ribatti,
Stefania Marsico,
Paola Apostoli,
Mark A. Slevin,
Marco Rusnati,
Carlos A. Guzman,
Simona Fiorentini,
Arnaldo Caruso
[show abstract]
[hide abstract]
ABSTRACT: Vascular diseases supported by aberrant angiogenesis have increased incidence in HIV-1–infected patients. Several data suggest
that endothelium dysfunction relies on action of HIV-1 proteins rather than on a direct effect of the virus itself. The HIV-1
matrix protein p17 is known to deregulate the biological activity of different immune cells. Recently, p17 was found to mimic
IL-8 chemokine activity by binding to the IL-8 receptor CXCR1. Here we show that p17 binds with high affinity to CXCR2, a
CXCR1-related receptor, and promotes the formation of capillary-like structures on human endothelial cells (ECs) by interacting
with both CXCR1 and CXCR2 expressed on the EC surface. ERK signaling via Akt was defined as the pathway responsible for p17-induced
tube formation. Ex vivo and in vivo experimental models confirmed the provasculogenic activity of p17, which was comparable
to that induced by VEGF-A. The hypothesis of a major role for p17 in HIV-1–induced aberrant angiogenesis is enforced by the
finding that p17 is detected, as a single protein, in blood vessels of HIV-1–patients and in particular in the nucleus of
ECs. Localization of p17 in the nucleus of ECs was evidenced also in in vitro experiments, suggesting the internalization
of exogenous p17 in ECs by mechanisms of receptor-mediated endocytosis. Recognizing p17 interaction with CXCR1 and CXCR2 as
the key event in sustaining EC aberrant angiogenesis could help us to identify new treatment strategies in combating AIDS-related
vascular diseases.
Proceedings of the National Academy of Sciences 09/2012; 109(36):14580-14585. · 9.68 Impact Factor
-
Augusto Pessina,
Valentina Coccè,
Arianna Bonomi,
Loredana Cavicchini,
Francesca Sisto,
Maura Ferrari,
Emilio Ciusani,
Stefania Navone,
Giovanni Marfia,
Eugenio Parati, Giulio Alessandri
[show abstract]
[hide abstract]
ABSTRACT: The main goal in cancer chemotherapy is to drive the drug into the tumor microenvironment to kill as many cancer cells as possible while producing the lowest collateral toxicity. Previously, we have shown that human bone marrow derived mesenchymal stromal cells (hBM-MSCs) exposed to Paclitaxel (PTX) were able to uptake and subsequently release the drug in the culture medium. PTX primed hBM-MSCs (hBM-MSCsPTX) located in the vicinity of cancer cells produced a strong inhibition of tumor cell growth both in vitro and in vivo. To expand these observations, in the present study we exposed human skin derived fibroblasts (hSDFs) to 2,000 ng/ml of PTX and then tested both cells and their conditioned medium (CM) in vitro for their capacity to inhibit the proliferation of human tumor cell lines (MOLT-4,DU-145,U87-MG,SH-SY5Y(+) and LAN-5). We found that hSDFs primed with PTX (hSDFsPTX) were able to uptake and subsequently release PTX in a time dependent manner. hSDFsPTX-derived CM(hSDFsPTX-CM) from 1:4 to 1:10 dilutions produced a significant (p<0.05) in vitro tumor growth inhibition. hSDFsPTX co-cultured with leukemia cells at 1:1 to 1:10 ratio, completely inhibited cells growth whereas no inhibition was induced by normal hSDFs cells. Our results demonstrate for the first time that hSDFs can be loaded in vitro with PTX and thus can acquire a potent anti-tumor activity. Since hSDFs can be easily isolated from skin biopsies without any particular pain and discomfort to donor patients, we conclude that hSDFs may represent a valid cell type option for carrying and delivering anti-cancer drugs.
Anti-cancer agents in medicinal chemistry 08/2012;
-
Piera Balzarini,
Anna Benetti,
Gloria Invernici,
Silvia Cristini,
Sonia Zicari,
Arnaldo Caruso,
Luisa B Gatta,
Angiola Berenzi,
Luisa Imberti,
Cinzia Zanotti,
Nazario Portolani,
Stefano M Giulini,
Maura Ferrari,
Emilio Ciusani,
Stefania E Navone,
Alessandra Canazza,
Eugenio A Parati, Giulio Alessandri
[show abstract]
[hide abstract]
ABSTRACT: Hepatocellular carcinoma (HCC) is a very angiogenic and malignant cancer. Conventional chemotherapy is poorly effective because of the abnormal structural organization of HCC-infiltrating vessels. In previous work, we demonstrated that HCC angiogenesis is driven by transforming growth factor beta-1(TGF-β1)/CD105 axis, stimulating liver-derived microvascular endothelial cells (Ld-MECs) migration. As TGF-β1 also affects mural cells (MCs) recruitment and maturation, we asked whether it may contribute to HCC-induced vascular abnormalities. HCC and adjacent non-neoplastic liver (nNL) biopsies obtained from 12 patients were analyzed by immunohistochemistry for angiogenic markers CD105, TGF-β1, CD44 and vascular endothelial growth factor-a (VEGFa) and for MC markers NG2, α-smooth muscle actin (αSMA) and neural cell adhesion molecule (NCAM). The same markers were also investigated by immunocytochemistry on cultured HCC-derived stromal cells (HCC-StCs) and nNL-derived StCs (nNL-StCs) isolated from the same liver biopsies. Angiogenic factors released by StCs were analyzed by ELISA and the interaction between StCs and Ld-MECs by adhesion assay. Compared with nNL, HCC biopsies showed increased angiogenic markers and αSMA that was localized in vessels. By contrast, NG2 and NCAM were substantially localized in tumor cells but absent in vessels and stroma. Cultured HCC-StCs showed less expression of NG2, αSMA and NCAM. They also demonstrated a lower capacity to release angiogenic factors and adhered on Ld-MECs. HCC-StCs and nNL-StCs treated with TGF-β1 or with of HepG2 (a human hepatoma cell line) derived conditioned medium (CM), down-modulated NCAM expression, whereas anti-NCAM antibodies significantly reduced the adhesion of StCs to Ld-MECs. By further blocking TGF-β1 with anti-TGF-β1 antibodies or with Ly-364947 (a specific inhibitor TGF-β1-receptor) adhesion to Ld-MECs and NCAM expression respectively was partially restored. TGF-β1 contributes to HCC-induced vascular alterations by affecting the interaction between HCC-StCs and Ld-MECs through a down-modulation of NCAM expression.
Laboratory Investigation 06/2012; 92(9):1297-309. · 3.64 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The expressions of p16, Ki-67, and L1 proteins and human papillomavirus DNA were investigated using polymerase chain reaction (HPV/PCR) and catalyzed signal-amplified colorimetric DNA in situ hybridization (CSAC/ISH) as potential molecular markers for the diagnosis and transforming potential of low cervical intraepithelial neoplasia (CIN1). Ki-67 and p16 protein expression increased linearly from control cases to more dysplastic cases (CIN1, CIN2, and CIN3), peaking in squamous cell carcinoma cases (P<0.05). In contrast, L1 expression was inversely correlated with malignant transformation. Patients with CIN1 were divided into 4 groups: L1p16, L1p16, L1p16, and L1p16, and the immunohistochemical results were combined with HPV/PCR, L1/PCR, and high-risk E6/E7 genome and CSAC/ISH data. Malignant transformation correlated with L1p16 patients (100% of CIN2, CIN3, and squamous cell carcinoma cases) and was evident in approximately 23% of CIN1 cases. In addition, the presence of HPV/DNA was evident in 52% of CIN1 cases, and within the L1p16 group. In 4 of 7 cases, the high-risk E6/E7 HPV genome was present and in 1 case it was integrated into the host DNA, as confirmed using CSAC/ISH. In patients with CIN1, investigating the presence of HPV/DNA using PCR and the presence of the high-risk E6/E7 genome is necessary to distinguish high-risk oncogenic patient groups from low-risk groups. This study highlights the importance of combining immunohistochemical analysis with HPV/PCR and CSAC/ISH to identify patients with CIN1 with a risk of neoplastic progression.
International journal of gynecological pathology: official journal of the International Society of Gynecological Pathologists 11/2011; 30(6):597-604. · 2.07 Impact Factor
-
Silvia Cristini, Giulio Alessandri,
Francesco Acerbi,
Emilio Ciusani,
Augusto Colombo,
Umberto Fascio,
Roberto F Nicosia,
Roberto W Invernizzi,
Maurizio Gelati,
Eugenio A Parati,
Gloria Invernici
[show abstract]
[hide abstract]
ABSTRACT: Complex microenvironmental stimuli influence neural cell properties. To study this, we developed a three-dimensional (3-D) neural culture system, composed of different populations including neurons, astrocytes, and neural stem cells (NSCs). In particular, these last-mentioned cells represent a source potentially exploitable to test drugs, to study neurodevelopment and cell-therapies for neuroregenerations. On seeding on matrigel in a medium supplemented with serum and mitogens, cells obtained from human fetal brain tissue formed 3-D self-organizing neural architectures. Immunocytochemical analysis demonstrated the presence of undifferentiated nestin+ and CD133+ cells, surrounded by β-tub-III+ and GFAP+ cells, suggesting the formation of niches containing potential human NSCs (hNSCs). The presence of hNSCs was confirmed by both neurosphere assay and RT-PCR, and their multipotentiality was demonstrated by both immunofluorescent staining and RT-PCR. Flow cytometry analysis revealed that neurosphere forming cells originating from at least two different subsets expressing, respectively, CD133 and CD146 markers were endowed with different proliferative and differentiation potential. Our data implicate that the complexity of environment within niches and aggregates of heterogeneous neural cell subsets may represent an innovative platform for neurobiological and neurodevelopmental investigations and a reservoir for a rapid expansion of hNSCs.
Tissue Engineering Part C Methods 07/2011; 17(11):1109-20. · 4.64 Impact Factor
-
Antonella Blasi,
Carmela Martino,
Luigi Balducci,
Marilisa Saldarelli,
Antonio Soleti,
Stefania E Navone,
Laura Canzi,
Silvia Cristini,
Gloria Invernici,
Eugenio A Parati, Giulio Alessandri
[show abstract]
[hide abstract]
ABSTRACT: Mesenchymal stem cells (MSCs) are multipotent stem cells able to differentiate into different cell lineages. However, MSCs represent a subpopulation of a more complex cell composition of stroma cells contained in mesenchymal tissue. Due to a lack of specific markers, it is difficult to distinguish MSCs from other more mature stromal cells such as fibroblasts, which, conversely, are abundant in mesenchymal tissue. In order to find more distinguishing features between MSCs and fibroblasts, we studied the phenotypic and functional features of human adipose-derived MSCs (AD-MSCs) side by side with normal human dermal fibroblasts (HNDFs) in vitro
AD-MSCs and HNDFs were cultured, expanded and phenotypically characterized by flow cytometry (FC). Immunofluorescence was used to investigate cell differentiation. ELISA assay was used to quantify angiogenic factors and chemokines release. Cultures of endothelial cells (ECs) and a monocyte cell line, U937, were used to test angiogenic and anti-inflammatory properties.
Cultured AD-MSCs and HNDFs display similar morphological appearance, growth rate, and phenotypic profile. They both expressed typical mesenchymal markers-CD90, CD29, CD44, CD105 and to a minor extent, the adhesion molecules CD54, CD56, CD106 and CD166. They were negative for the stem cell markers CD34, CD146, CD133, CD117. Only aldehyde dehydrogenase (ALDH) was expressed. Neither AD-MSCs nor HNDFs differed in their multi-lineage differentiation capacity; they both differentiated into osteoblast, adipocyte, and also into cardiomyocyte-like cells. In contrast, AD-MSCs, but not HNDFs, displayed strong angiogenic and anti-inflammatory activity. AD-MSCs released significant amounts of VEGF, HGF and Angiopoietins and their conditioned medium (CM) stimulated ECs proliferation and tube formations. In addition, CM-derived AD-MSCs (AD-MSCs-CM) inhibited adhesion molecules expression on U937 and release of RANTES and MCP-1. Finally, after priming with TNFα, AD-MSCs enhanced their anti-inflammatory potential; while HNDFs acquired pro-inflammatory activity.
AD-MSCs cannot be distinguished from HNDFs in vitro by evaluating their phenotypic profile or differentiation potential, but only through the analysis of their anti-inflammatory and angiogenic properties. These results underline the importance of evaluating the angiogenic and anti-inflammatory features of MSCs preparation. Their priming with inflammatory cytokines prior to transplantation may improve their efficacy in cell-based therapies for tissue regeneration.
Vascular cell. 02/2011; 3(1):5.
-
[show abstract]
[hide abstract]
ABSTRACT: Primary malignant central nervous system (CNS) tumors only represent about 2% of all cancers. However, they are very often associated with high morbidity and mortality. Despite current standard-of-care therapy, such as surgery, irradiation, and chemotherapy, neither cure nor any toxic therapy against malignant CNS tumors has been developed so far. Nanotechnology may alter this situation. It offers a new promise for cancer diagnosis and treatment. This emerging technology, by developing and manufacturing materials using atomic and molecular elements, can provide a platform for the combination of diagnostics, therapeutics and delivery to the tumor, with subsequent monitoring of the response. This review focuses on recent developments in cancer nanotechnology with particular attention to nanoparticle systems, important tools for the improvement of drug delivery in brain tumor. The latest advances in both the research sector and in recent patents for cancer imaging and therapy are discussed.
Recent patents on anti-cancer drug discovery. 01/2011; 6(1):58-69.
-
Augusto Pessina,
Arianna Bonomi,
Valentina Coccè,
Gloria Invernici,
Stefania Navone,
Loredana Cavicchini,
Francesca Sisto,
Maura Ferrari,
Lucia Viganò,
Alberta Locatelli,
Emilio Ciusani,
Graziella Cappelletti,
Daniele Cartelli,
Caruso Arnaldo,
Eugenio Parati,
Giovanni Marfia,
Roberto Pallini,
Maria Laura Falchetti, Giulio Alessandri
[show abstract]
[hide abstract]
ABSTRACT: Mesenchymal stromal cells may represent an ideal candidate to deliver anti-cancer drugs. In a previous study, we demonstrated that exposure of mouse bone marrow derived stromal cells to Doxorubicin led them to acquire anti-proliferative potential towards co-cultured haematopoietic stem cells (HSCs). We thus hypothesized whether freshly isolated human bone marrow Mesenchymal stem cells (hMSCs) and mature murine stromal cells (SR4987 line) primed in vitro with anti-cancer drugs and then localized near cancer cells, could inhibit proliferation.
Paclitaxel (PTX) was used to prime culture of hMSCs and SR4987. Incorporation of PTX into hMSCs was studied by using FICT-labelled-PTX and analyzed by FACS and confocal microscopy. Release of PTX in culture medium by PTX primed hMSCs (hMSCsPTX) was investigated by HPLC. Culture of Endothelial cells (ECs) and aorta ring assay were used to test the anti-angiogenic activity of hMSCsPTX and PTX primed SR4987(SR4987PTX), while anti-tumor activity was tested in vitro on the proliferation of different tumor cell lines and in vivo by co-transplanting hMSCsPTX and SR4987PTX with cancer cells in mice. Nevertheless, despite a loss of cells due to chemo-induced apoptosis, both hMSCs and SR4987 were able to rapidly incorporate PTX and could slowly release PTX in the culture medium in a time dependent manner. PTX primed cells acquired a potent anti-tumor and anti-angiogenic activity in vitro that was dose dependent, and demonstrable by using their conditioned medium or by co-culture assay. Finally, hMSCsPTX and SR4987PTX co-injected with human cancer cells (DU145 and U87MG) and mouse melanoma cells (B16) in immunodeficient and in syngenic mice significantly delayed tumor takes and reduced tumor growth.
These data demonstrate, for the first time, that without any genetic manipulation, mesenchymal stromal cells can uptake and subsequently slowly release PTX. This may lead to potential new tools to increase efficacy of cancer therapy.
PLoS ONE 01/2011; 6(12):e28321. · 4.09 Impact Factor
-
Rocco De Siena,
Luigi Balducci,
Antonella Blasi,
Manuela Gessica Montanaro,
Marilisa Saldarelli,
Vittorio Saponaro,
Carmela Martino,
Gaetano Logrieco,
Antonio Soleti,
Simona Fiobellot,
Paolo Madeddu,
Giacomo Rossi,
Domenico Ribatti,
Antonio Crovace,
Silvia Cristini,
Gloria Invernici,
Eugenio Agostino Parati, Giulio Alessandri
[show abstract]
[hide abstract]
ABSTRACT: Cell-based therapy could be a valid option to treat myocardial infarct (MI). Adipose-derived stromal cells (ADStCs) have demonstrated tissue regenerative potential including cardiomyogenesis. Omentum is an extremely rich source of visceral fat and its accumulation seems to correlate with cardiovascular diseases. We investigated the capacity of human fat Omentum-derived StCs (FOStCs) to affect heart function upon acute infarct in pigs induced by permanent ligation of the anterior interventricular artery (IVA). We demonstrated for the first time that the local injection of 50x10(6) of FOStCs ameliorates the functional parameters of post-infarct heart. Most importantly, histology of FOStCs treated hearts demonstrated a substantial improvement of cardiomyogenesis. In culture, FOStCs produced an impressive number and amount of angiogenic factors and cytokines. Moreover, the conditioned medium of FOStCs (FOStCs-CM) stimulates in vitro cardiac endothelial cells (ECs) proliferation and vascular morphogenesis and inhibits monocytes, EC activation and cardiomyocyte apoptosis. Since FOStCs in vivo did not trans-differentiate into cardiomyocyte-like cells, we conclude that FOStCs efficacy was presumably mediated by a potent paracrine mechanism involving molecules that concomitantly improved angiogenesis, reduced inflammation and prevented cardiomyocytes death. Our results highlight for the first time the important role that human FOStCs may have in cardiac regeneration.
Experimental Cell Research 02/2010; 316(11):1804-15. · 3.58 Impact Factor
-
Silvia Cristini,
Stefania Navone,
Laura Canzi,
Francesco Acerbi,
Emilio Ciusani,
Uros Hladnik,
Paola de Gemmis, Giulio Alessandri,
Augusto Colombo,
Eugenio Parati,
Gloria Invernici
[show abstract]
[hide abstract]
ABSTRACT: The study of Lesch-Nyhan-diseased (LND) human brain is crucial for understanding how mutant hypoxanthine-phosphoribosyltransferase (HPRT) might lead to neuronal dysfunction. Since LND is a rare, inherited disorder caused by a deficiency of the enzyme HPRT, human neural stem cells (hNSCs) that carry this mutation are a precious source for delineating the consequences of HPRT deficiency and for developing new treatments. In our study we have examined the effect of HPRT deficiency on the differentiation of neurons in hNSCs isolated from human LND fetal brain. We have examined the expression of a number of transcription factors essential for neuronal differentiation and marker genes involved in dopamine (DA) biosynthetic pathway. LND hNSCs demonstrate aberrant expression of several transcription factors and DA markers. HPRT-deficient dopaminergic neurons also demonstrate a striking deficit in neurite outgrowth. These results represent direct experimental evidence for aberrant neurogenesis in LND hNSCs and suggest developmental roles for other housekeeping genes in neurodevelopmental disease. Moreover, exposure of the LND hNSCs to retinoic acid medium elicited the generation of dopaminergic neurons. The lack of precise understanding of the neurological dysfunction in LND has precluded development of useful therapies. These results evidence aberrant neurogenesis in LND hNSCs and suggest a role for HPRT gene in neurodevelopment. These cells combine the peculiarity of a neurodevelopmental model and a human, neural origin to provide an important tool to investigate the pathophysiology of HPRT deficiency and more broadly demonstrate the utility of human neural stem cells for studying the disease and identifying potential therapeutics.
Human Molecular Genetics 02/2010; 19(10):1939-50. · 7.64 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Human herpesvirus 6 (HHV-6) is a lymphotropic virus, but recent observations showed that also vascular endothelial cells (ECs) are susceptible to infection, both in vivo and in vitro. The observation that lymph nodes are a site of viral persistence suggests that lymphatic ECs (LECs) might be even more relevant for HHV-6 biology than vascular ECs. Here, we provide evidence that HHV-6 can infect LECs in vitro and establish a latent infection. Thus HHV-6 infection induces the loss of angiogenic properties both in LECs and in vascular ECs, as shown by the inability to form capillary-like structures and to seal wound scratches. The antiangiogenic effects observed in infected cells are associated to the expression of HHV-6 U94/rep, a latency-associated gene. In fact, transfection of U94/rep or addition of recombinant U94/REP protein to ECs inhibits the formation of in vitro capillary-like structures, reduces migration of ECs, and blocks angiogenesis, rendering rat aortic rings insensitive to VEGF-induced vasculogenetic activity. The ability of U94/rep to block different angiogenetic steps may lead to approaches in the potential control of the proliferation of blood and lymphatic vessels.
Proceedings of the National Academy of Sciences 11/2009; 106(48):20446-51. · 9.68 Impact Factor
-
Lucíola S Barcelos,
Cécile Duplaa,
Nicolle Kränkel,
Gallia Graiani,
Gloria Invernici,
Rajesh Katare,
Mauro Siragusa,
Marco Meloni,
Ilaria Campesi,
Manuela Monica,
Andreas Simm,
Paola Campagnolo,
Giuseppe Mangialardi,
Lara Stevanato, Giulio Alessandri,
Costanza Emanueli,
Paolo Madeddu
[show abstract]
[hide abstract]
ABSTRACT: We evaluated the healing potential of human fetal aorta-derived CD133(+) progenitor cells and their conditioned medium (CD133(+) CCM) in a new model of ischemic diabetic ulcer. Streptozotocin-induced diabetic mice underwent bilateral limb ischemia and wounding. One wound was covered with collagen containing 2x10(4) CD133(+) or CD133(-) cells or vehicle. The contralateral wound, covered with only collagen, served as control. Fetal CD133(+) cells expressed high levels of wingless (Wnt) genes, which were downregulated following differentiation into CD133(-) cells along with upregulation of Wnt antagonists secreted frizzled-related protein (sFRP)-1, -3, and -4. CD133(+) cells accelerated wound closure as compared with CD133(-) or vehicle and promoted angiogenesis through stimulation of endothelial cell proliferation, migration, and survival by paracrine effects. CD133(+) cells secreted high levels of vascular endothelial growth factor (VEGF)-A and interleukin (IL)-8. Consistently, CD133(+) CCM accelerated wound closure and reparative angiogenesis, with this action abrogated by co-administering the Wnt antagonist sFRP-1 or neutralizing antibodies against VEGF-A or IL-8. In vitro, these effects were recapitulated following exposure of high-glucose-primed human umbilical vein endothelial cells to CD133(+) CCM, resulting in stimulation of migration, angiogenesis-like network formation and induction of Wnt expression. The promigratory and proangiogenic effect of CD133(+) CCM was blunted by sFRP-1, as well as antibodies against VEGF-A or IL-8. CD133(+) cells stimulate wound healing by paracrine mechanisms that activate Wnt signaling pathway in recipients. These preclinical findings open new perspectives for the cure of diabetic ulcers.
Circulation Research 05/2009; 104(9):1095-102. · 9.49 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Vasculogenesis, the formation of blood vessels in embryonic or fetal tissue mediated by immature vascular cells (i.e., angioblasts), is poorly understood. Here we report a summary of our recent studies on the identification of a population of vascular progenitor cells (VPCs) in human fetal aorta. These undifferentiated mesenchymal cells co-express endothelial and myogenic markers (CD133+, CD34+, KDR+, desmin+) and are localized in outer layer of the aortic stroma of 11-12 weeks old human fetuses. Under stimulation with VEGF-A or PDGF-BB, VPCs give origin to a mixed population of mature endothelial and mural cells, respectively. When embedded in a three-dimensional collagen gel, VPCs organize into cohesive cellular cords that resembled mature vascular structures. The therapeutic efficacy of a small number of VPCs transplanted into ischemic limb muscle was demonstrated in immunodeficient mice. Investigation of the effect of VPCs on experimental heart ischemia and on diabetic ischemic ulcers in mice is in progress and seems to confirm their efficacy. On the whole, fetal aorta represents an important source for the investigation of phenotypic and functional features of human vascular progenitor cells.
Cytotechnology 11/2008; 58(1):43-7. · 1.21 Impact Factor
-
Anna Benetti,
Angiola Berenzi,
Marco Gambarotti,
Emirena Garrafa,
Maurizio Gelati,
Enrico Dessy,
Nazario Portolani,
Tullio Piardi,
Stefano Maria Giulini,
Arnaldo Caruso,
Gloria Invernici,
Eugenio Agostino Parati,
Roberto Nicosia, Giulio Alessandri
[show abstract]
[hide abstract]
ABSTRACT: 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.
Cancer Research 11/2008; 68(20):8626-34. · 7.86 Impact Factor
-
Maria Laura Falchetti,
Maria Patrizia Mongiardi,
Paolo Fiorenzo,
Giovanna Petrucci,
Francesco Pierconti,
Igea D'Agnano,
Giorgio D'Alessandris, Giulio Alessandri,
Maurizio Gelati,
Lucia Ricci-Vitiani,
Giulio Maira,
Luigi Maria Larocca,
Andrea Levi,
Roberto Pallini
[show abstract]
[hide abstract]
ABSTRACT: Tumor angiogenesis is a complex process that involves a series of interactions between tumor cells and endothelial cells (ECs). In vitro, glioblastoma multiforme (GBM) cells are known to induce an increase in proliferation, migration and tube formation by the ECs. We have previously shown that in human GBM specimens the proliferating ECs of the tumor vasculature express the catalytic component of telomerase, hTERT, and that telomerase can be upregulated in human ECs by exposing these cells to GBM in vitro. Here, we developed a controlled in vivo assay of tumor angiogenesis in which primary human umbilical vascular endothelial cells (HUVECs) were subcutaneously grafted with or without human GBM cells in immunocompromised mice as Matrigel implants. We found that primary HUVECs did not survive in Matrigel implants, and that telomerase upregulation had little effect on HUVEC survival. In the presence of GBM cells, however, the grafted HUVECs not only survived in Matrigel implants but developed tubule structures that integrated with murine microvessels. Telomerase upregulation in HUVECs enhanced such effect. More importantly, inhibition of telomerase in HUVECs completely abolished tubule formation and greatly reduced survival of these cells in the tumor xenografts. Our data demonstrate that telomerase upregulation by the ECs is a key requisite for GBM tumor angiogenesis.
International Journal of Cancer 04/2008; 122(6):1236-42. · 5.44 Impact Factor
-
Gloria Invernici,
Silvia Cristini,
Paolo Madeddu,
Stefano Brock,
Frank Spillmann,
Pia Bernasconi,
Cristina Cappelletti,
Chiara Calatozzolo,
Umberto Fascio,
Gianluigi Bisleri,
Claudio Muneretto, Giulio Alessandri,
Eugenio A Parati
[show abstract]
[hide abstract]
ABSTRACT: Cell transplantation to repair or regenerate injured myocardium is a new frontier in the treatment of cardiovascular disease. Most studies on stem cell transplantation therapy in both experimental heart infarct and in phase-I human clinical trials have focused on the use of undifferentiated stem cells. Based on our previous observations demonstrating the presence of multipotent progenitor cells in human adult skeletal muscle, in this study we investigated the capacity of these progenitors to differentiate into cardiomyocytes. Here we show an efficient protocol for the cardiomyogenic differentiation of human adult skeletal muscle stem cells in vitro. We found that treatment with Retinoic Acid directed cardiomyogenic differentiation of skeletal muscle stem cells in vitro. After Retinoic Acid treatment, cells expressed cardiomyocyte markers and acquired spontaneous contraction. Functional assays exhibited cardiac-like response to increased extracellular calcium. When cocultured with mouse cardiomyocytes, Retinoic Acid-treated skeletal muscle stem cells expressed connexin43 and when transplanted into ischemic heart were detectable even 5 weeks after injection. Based on these results, we can conclude that human adult skeletal muscle stem cells, if opportunely treated, can transdifferentiate into cells of cardiac lineage and once injected into infarcted heart can integrate, survive in cardiac tissue and improve the cardiac function.
Experimental Cell Research 02/2008; 314(2):366-76. · 3.58 Impact Factor
-
Gloria Invernici,
Costanza Emanueli,
Paolo Madeddu,
Silvia Cristini,
Sergio Gadau,
Anna Benetti,
Emilio Ciusani,
Giorgio Stassi,
Mauro Siragusa,
Roberto Nicosia,
Cesare Peschle,
Umberto Fascio,
Augusto Colombo,
Tommaso Rizzuti,
Eugenio Parati, Giulio Alessandri
[show abstract]
[hide abstract]
ABSTRACT: Vasculogenesis, the formation of blood vessels in embryonic or fetal tissue mediated by immature vascular cells (ie, angioblasts), is poorly understood. We report the identification of a population of vascular progenitor cells (hVPCs) in the human fetal aorta composed of undifferentiated mesenchymal cells that coexpress endothelial and myogenic markers. Under culture conditions that promoted cell differentiation, hVPCs gave rise to a mixed population of mature endothelial and mural cells when progenitor cells were stimulated with vascular endothelial growth factor-A or platelet-derived growth factor-betabeta. hVPCs grew as nonadherent cells and, when embedded in a three-dimensional collagen gel, reorganized into cohesive cellular cords that resembled mature vascular structures. hVPC-conditioned medium contained angiogenic substances (vascular endothelial growth factor-A and angiopoietin-2) and strongly stimulated the proliferation of endothelial cells. We also demonstrate the therapeutic efficacy of a small number of hVPCs transplanted into ischemic limb muscle of immunodeficient mice. hVPCs markedly improved neovascularization and inhibited the loss of endogenous endothelial cells and myocytes, thus ameliorating the clinical outcome from ischemia. We conclude that fetal aorta represents an important source for the investigation of the phenotypic and functional features of human vascular progenitor cells.
American Journal Of Pathology 07/2007; 170(6):1879-92. · 4.89 Impact Factor
-
Elena Monzani,
Floriana Facchetti,
Enrico Galmozzi,
Elena Corsini,
Anna Benetti,
Chiara Cavazzin,
Angela Gritti,
Andrea Piccinini,
Danilo Porro,
Mario Santinami,
Gloria Invernici,
Eugenio Parati, Giulio Alessandri,
Caterina A M La Porta
[show abstract]
[hide abstract]
ABSTRACT: The failure to eradicate most cancers and in particular melanoma may be as fundamental as a misidentification of the target. The identification of cancer stem/initiating cells within the tumour population with a crucial role for tumour formation may open new pharmacological perspectives. Our data show three main novelties for human melanoma: firstly, melanoma biopsy contains a subset of cells expressing CD133 (CD133+) and the latter is able to develop a Mart-1 positive tumour in NOD-SCID mice. Secondly, the WM115, a human melanoma cell line, has been found to express both CD133 and ABCG2 markers. This cell line grows as floating spheroids, expresses typical progenitors and mature neuronal/oligodendrocyte markers and is able to transdifferentiate into astrocytes or mesenchymal lineages under specific growth conditions. As in xenografts generated with CD133+ biopsy melanoma cells, those produced by the cell line displayed lower levels of CD133 and ABCG2. Thirdly, the WM115 cells express the most important angiogenic and lymphoangiogenic factors such as notch 4, prox1 and podoplanin which can cooperate in the development of the tumourigenic capability of melanoma in vivo. Therefore, in this study, we demonstrate the presence of stem/initiating subsets in melanoma both in biopsy and in an established melanoma cell line grown in vitro and in xenografts. Interestingly, considering that melanoma gives metastasis primarily through lymphatic vessels, herein, we demonstrated that a melanoma cell line expresses typical lymphoangiogenic factors.
European Journal of Cancer 04/2007; 43(5):935-46. · 5.54 Impact Factor
