Carmen-Mariana AANEI

Centre Hospitalier Universitaire de Saint-Etienne, Saint Etienne, .

E-mail: trimite un mesaj.

Nascut(a) in: 1972

Interese: celulele stromale mezenchimale, sindroame mielodisplazice, proteinele de adeziune focala

Detalii:
Studiile pe care le-am realizat au avut drept scop intelegerea rolului micromediului medular in patogeneza sindroamelor mielodisplazice, tinandu-se cont de faptul ca celulele stromale mezenchimale (CSM), componente principale ale micromediului, mediaza contactele directe cu celulele precursoare hematopoietice (CPH).
Obiectivele tezei mele de doctorat au fost: de a evalua deficientele de crestere ale CSM izolate de la pacientii cu sindroame mielodisplazice (SMD)prin comparatie cu celulele normale izolate de la donatorii sanatosi, de a explora profilul lor de aderenta, de a identifica substraturile moleculare ale aderentelor focale (AF) pe care CSM le formeaza si, in final de a realiza corelatii intre aceste proteine cu anomaliile functionale ale CSM, precum si cu disfunctiile de crestere identificate in compartimentul hematopoietic conex. Pentru inceput am imaginat un protocol de selectie imunomagnetica a CSM bazat pe expresia a doi marcheri specifici, STRO-1 si CD73, in scopul de a obtine preparate celulare conforme exigentelor cerute de Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy. Apoi am evaluat profilul lor de adeziune si caracteristicile lor de crestere in cultura (comparand fractiile izolate de la pacientii cu SMD cu celulele normale). Testele functionale au demonstrat ca CSM ale pacientilor cu SMD prezinta un declin continuu al proliferarii si capacitatii clonogenice pe parcursul celor 14 zile de cultura, in absenta oricaror semnalizari din partea CPH. Deficientele de crestere ale CSM s-au corelat semnificativ cu descresterea expresiei a doua molecule de adeziune CD44 si CD49e(α5-integrina). In plus si potentialul clonogenic al CPH, care este mediat prin contacte directe cu stroma a fost afectat prin scaderea expresiei moleculei CD49e. In final am realizat experimente de imunofluorescenta pentru a evalua expresia a doua proteine AF, paxilina si pFAK [Y397], precum si a doua molecule care controleaza activitatea acestora, HSP90αβ si p130CAS la nivelul CSM. Analiza acestor proteine a relevat faptul ca exista diferente calitative si cantitative de expresie a acestor proteine intre stroma displazica si cea normala, iar analiza colocalizarii a aratat o puternica asociere a paxilinei, pFAK si HSP90αβ la nivel nuclear in CSM patologice. Expresia crescuta si puternica colocalizare nucleara a proteinelor AF la HSP90αβ s-au corelat cu un avantaj proliferativ important al CSM selectate de la pacientii cu anemii refractare cu exces de blasti (AREB)si cu diminuarea capacitatii clonogenice a CPH. Aceste rezultate deschid oportunitati interesante pentru terapia SMD daca se tine cont de faptul ca proteinele AF intervin in interactiunile CPH-CSM si ca molecula FAK este o proteina client a HSP90αβ, toate acestea sprijinind utilizarea inhibitorilor de HSP90αβ in tratamentul adjuvant al SMD.

Rezultatele acestui studiu au fost diseminate in doua publicatii relevante pentru domeniu:
 Experimental Cell Research (Carmen Mariana Aanei, Florin Zugun Eloae, Pascale Flandrin, Emmanuelle Tavernier, Eugen Carasevici, Denis Guyotat, Lydia Campos, Focal adhesion protein abnormalities in myelodysplastic mesenchymal stromal cells, Experimental Cell Research 3 1 7 (2 0 1 1); 2 6 1 6 – 2 6 2 9 [PubMed : 21871449]),

 and Stem Cells and Development (Carmen Mariana Aanei, Pascale Flandrin, Florin Zugun Eloae, Eugen Carasevici, Denis Guyotat, Eric Wattel, Lydia Campos, Intrinsic growth deficiencies of mesenchymal stromal cells (MSCs) in myelodysplastic syndromes (MDS), Stem Cells and Development (2012); 21(10):1604-15 [PubMed: 21933023 ]). Acest articol a fost desemnat intre primele cinci "High Impact Articles" ale revistei Stem Cells and Development review (July issue).
In plus aceste articole au fost deja citate de o recenzie asupra anomaliilor intalnite la nivelul stromei in SMD (Raaijmakers, Int J Hematol 2012)cat si de alte doua articole din domeniu Santamaria, Haematologica 2012 si Susanta K. Hui, PLoS ONE 2012.
De asemenea, rezultatele au fost prezentate la diverse evenimente stiintifice. Ultimul a fost AACR Annual Meeting 2012 unde American Association for Cancer Research Committee mi-a acordat distinctia "Highly Rated Paper" pentru abstractul "Adhesion-mediated dysfunctions in myelodysplastic syndromes microenvironment” care a fost cotat intre primele 2.5% din 5757 inregistrate in competitie.

Details:
The studies that I have conducted are related to understanding the place of stromal microenvironment in myelodysplastic syndromes (MDS) pathophysiology which mediates the direct contact with haematopoietic precursor cells (HPCs).
The aims of my PhD thesis were to evaluate the putative growth deficiencies of mesenchymal stromal cells (MSC) from MDS individuals compared with normal controls, to explore their adhesion profile, and further, to assess the molecular substrates involved in the focal adherences that they form, and finally, to perform correlations with their growth dysfunctions and with haematopoietic compartment abnormalities.
To this end, I developed a novel selection protocol in order to obtain a standards-compliant MSC preparation using their different expression for the specific markers STRO-1 and CD73.
Thereafter, I evaluated their adhesion phenotype and the growth patterns of these MSC selected fractions from MDS patients vs. normal controls. Functional assays revealed that the MSCs from MDS are intrinsically pathological, showing a continuous decline of proliferation and a reduced clonogenic capacity during 14 days of culture and in the absence of signals from hematopoietic cells. The MSC growth defects were significantly correlated with decreases in CD44 adhesion molecules and CD49e (α5-integrin). Furthermore, the clonogenic potential of HPC is controlled by adhesion mechanisms dependent on stroma, and α5-integrin might be one of the molecules involved in this process.
Finally, I conducted immunofluorescence experiments to assess two focal adhesion proteins paxillin, and pFAK [Y397], and two regulatory proteins, HSP90αβ, and p130CAS in terms of reactivity, intensity and cellular localization. Immunofluorescence microscopy allowed the identification of qualitative and quantitative differences, and subcellular localization analysis revealed that, in pathological MSCs, paxillin, pFAK, and HSP90αβ form nuclear, molecular complexes. An increased
expression of paxillin, pFAK [Y397], and HSP90αβ, in addition to, their stronger nuclear colocalization, correlated with a consistent proliferative advantage in MSCs from Refractory Anaemia with Excess Blasts (RAEB) and has been shown to have a negative impact on clonogenic capacity of HPCs.
These results open interesting opportunities, e.g. signalling via FA proteins could be implicated in HPC-to-MSC interactions, and, considering that FAK is one of HSP90αβ-client proteins, enhance the utility of HSP90αβ inhibition as a target for adjuvant myelodysplasia therapy.

The results of this study have disseminated in two highly relevant publications, in the field:

 Experimental Cell Research (Carmen Mariana Aanei, Florin Zugun Eloae, Pascale Flandrin, Emmanuelle Tavernier, Eugen Carasevici, Denis Guyotat, Lydia Campos, Focal adhesion protein abnormalities in myelodysplastic mesenchymal stromal cells, Experimental Cell Research 3 1 7 (2 0 1 1); 2 6 1 6 – 2 6 2 9 [PubMed : 21871449]),

 and Stem Cells and Development (Carmen Mariana Aanei, Pascale Flandrin, Florin Zugun Eloae, Eugen Carasevici, Denis Guyotat, Eric Wattel, Lydia Campos, Intrinsic growth deficiencies of mesenchymal stromal cells (MSCs) in myelodysplastic syndromes (MDS), Stem Cells and Development (2012); 21(10):1604-15 [PubMed: 21933023 ]). This article has designated among the top five High Impact Articles from Stem Cells and Development review (July issue).

In adittion, these papers have already cited in a recent review on stromal anomalies in MDS (Raaijmakers, Int J Hematol 2012) and by two articles, one about the cytogenetic modifications of mesenchymal cells selected from MDS patients (Santamaria, Haematologica 2012)and second relevant for the utility of MSC in biotechnologies(Susanta K. Hui, PLoS ONE 2012).

Likewise, the results have communicated in several prestigious scientific meetings. The last one was AACR Annual Meeting 2012, where the American Association for Cancer Research Committee has designated my abstract “Adhesion-mediated dysfunctions in myelodysplastic syndromes microenvironment” as a Highly Rated Paper (abstract scored in the top 2.5% of abstracts presented in the poster sessions).

Publicații selectate:

* L. Campos, F. Solly, C. Aanei, P. Flandrin, D. Guyotat, HSP90 is overexpressed in high-risk myelodysplastic syndromes and associated with higher expression and activation of FAK, Leukemia Research, 33, 2009.

* Lydia Campos, Nathalie Nadal, Pascale Flandrin-Gresta, Christian Vasselon, Carmen Aanei, Claire Berger, and Jean Louis Stephan, Congenital Acute Leukemia with Initial Indolent Presentation—A Case Report, Cytometry B Clin Cytom, 80(2), 2011.

* Carmen Mariana Aanei, Florin Zugun Eloae, Pascale Flandrin, Emmanuelle Tavernier, Eugen Carasevici, Denis Guyotat, Lydia Campos, Focal adhesion protein abnormalities in myelodysplastic mesenchymal stromal cells, Experimental Cell Research, 3 1 7, 2011.

* Carmen Mariana Aanei, Pascale Flandrin, Florin Zugun Eloae, Eugen Carasevici, Denis Guyotat, Eric Wattel, Lydia Campos, Intrinsic growth deficiencies of mesenchymal stromal cells (MSCs) in myelodysplastic syndromes (MDS), Stem Cells and Development, 21(10), 2012.