Résumé : Les maladies cardiovasculaires représentent la principale cause de mortalité mondiale, soit le tiers des décès annuels selon l’Organisation mondiale de la Santé. L’hypercholestérolémie, caractérisée par une élévation des niveaux plasmatiques de lipoprotéines de faible densité (LDL), est l’un des facteurs de risque majeur pour les maladies cardiovasculaires. La proprotéine convertase subtilisine/kexine type 9 (PCSK9) joue un rôle essentiel dans l’homéostasie du cholestérol sanguin par la régulation des niveaux protéiques du récepteur LDL (LDLR). PCSK9 est capable de se lier au LDLR et favorise l’internalisation et la dégradation du récepteur dans les lysosomes. L’inhibition de PCSK9 s’avère une cible thérapeutique validée pour le traitement de l’hypercholestérolémie et la prévention des maladies cardiovasculaires. Par contre, plusieurs mécanismes responsables de la régulation et la dégradation du complexe PCSK9-LDLR n’ont pas encore été complètement caractérisés comme la régulation par la protéine annexin A2 (AnxA2), un inhibiteur endogène de PCSK9. De plus, plusieurs évidences suggèrent la présence d’une ou plusieurs protéines, encore inconnues, impliquées dans le mécanisme d’action de PCSK9. Celles-ci pourraient réguler l’internalisation et le transport du complexe PCSK9-LDLR vers les lysosomes. Les objectifs de cette thèse sont de mieux définir le rôle et l’impact de l’AnxA2 sur la protéine PCSK9 en plus d’identifier de nouveaux partenaires d’interactions de PCSK9 pour mieux caractériser son mécanisme d’action sur la régulation des niveaux de LDLR. Nous avons démontré que l’inhibition de PCSK9 par l’AnxA2 extracellulaire s’effectue via sa liaison aux domaines M1+M2 de la région C-terminale de PCSK9 et nous avons mis en évidence les premières preuves d’un contrôle intracellulaire de l’AnxA2 sur la traduction de l’ARNm de PCSK9. Nos résultats révèlent une liaison de l’AnxA2 à l’ARN messager de PCSK9 qui cause une répression traductionnelle. Nous avons également identifié la protéine glypican-3 (GPC3) comme un nouveau partenaire d’interaction extracellulaire avec le PCSK9 et intracellulaire avec le complexe PCSK9-LDLR dans le réticulum endoplasmique des cellules HepG2 et Huh7. Nos études démontrent que GPC3 réduit l’activité extracellulaire de PCSK9 en agissant comme un compétiteur du LDLR pour la liaison avec PCSK9. Une meilleure compréhension des mécanismes de régulation et de dégradation du complexe PCKS9-LDLR permettra de mieux évaluer l’impact et l’efficacité des inhibiteurs de la protéine PCSK9. / Abstract : Cardiovascular disease is the leading cause of global mortality, responsible for one third of global deaths, according to the latest statistics from World Health Organization. Hypercholesterolemia, characterized by increased plasma low-density lipoprotein (LDL) cholesterol, is a major determinant of cardiovascular disease risk. Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a critical role in cholesterol homeostasis by regulating LDL receptor (LDLR) protein levels. PCSK9 binds to the LDLR and promotes its internalization and degradation in late endosomal/lysosomal compartments. Inhibition of PCSK9 action on LDLR has emerged as a novel therapeutic target for hypercholesterolemia and the prevention of cardiovascular disease. Annexin A2 (AnxA2) was reported as an endogenous extracellular inhibitor of PCSK9 activity upon cell-surface LDLR degradation and mechanisms of PCSK9’s regulation by AnxA2. However, its role on PCSK9 regulation still need better characterization in hepatocellular carcinoma cell lines. Moreover, many evidences suggest the presence of additional unknown interaction partners involve in the LDLR regulation and degradation mediated by PCSK9. These unknown partners could regulate the internalization and trafficking of the PCSK9-LDLR complex to lysosomes. The objectives of this thesis are to better define the role and impact of AnxA2 on PCSK9 and to identify novel PCSK9 interacting partners that participate and regulate the PCSK9-LDLR complex formation and degradation. We demonstrated that PCSK9 inhibition by extracellular AnxA2 occurs via its interaction with the M1+M2 modules of PCSK9’s C-terminal region. Most importantly, we revealed a new role of intracellular AnxA2 in the reduction of PCSK9 protein levels via a translational mechanism. Our results suggest a translational repression from the binding of AnxA2 to PCSK9’s mRNA. Also, we successfully identified a novel and functional interaction between glypican-3 (GPC3) and PCSK9. We demonstrated the extracellular GPC3 interaction with PCSK9 and the intracellular GPC3 with both PCSK9 and LDLR in human hepatocellular carcinoma cell lines HepG2 and Huh7. Our studies revealed that extracellular GPC3 can act as an endogenous competitive binding partner of PCSK9 to the LDLR, and hence reducing its activity towards LDLR degradation. The continued understanding of PCSK9 interactions is critical, from a mechanistic point of view as well as from the optimization of therapeutic interventions.

Glypican-3 (GPC3) belongs to a family of cell surface proteoglycans. GPC3 regulates the activity of several morphogens and growth factors that play critical roles during development. Disrupting the function of GPC3 leads to disease, including the overgrowth disease Simpson Golabi Behmel Syndrome (SGBS) and Cancer. Previous work has shown that GPC3 is over expressed in Hepatocellular Carcinoma (HCC), and that HCC proliferation is stimulated through GPC3 mediated activation of the Wnt signaling pathway. Glypicans are known to regulate Wnt signaling in a variety of model organisms including Drosophila and mouse. This work investigates the hypothesis that GPC3 stimulates Wnt signaling by facilitating/stabilizing the interaction between Wnt and its receptor Frizzled (Fzd). Consistent with this hypothesis, we found that GPC3 is able to bind both Wnt and Fzd. The binding of GPC3 to Fzd is mediated by the GPC3 glycosaminoglycan chains and by the cysteine rich domain of Fzd.

Glypican-3 (GPC3) belongs to a family of cell surface proteoglycans. GPC3 regulates the activity of several morphogens and growth factors that play critical roles during development. Disrupting the function of GPC3 leads to disease, including the overgrowth disease Simpson Golabi Behmel Syndrome (SGBS) and Cancer. Previous work has shown that GPC3 is over expressed in Hepatocellular Carcinoma (HCC), and that HCC proliferation is stimulated through GPC3 mediated activation of the Wnt signaling pathway. Glypicans are known to regulate Wnt signaling in a variety of model organisms including Drosophila and mouse. This work investigates the hypothesis that GPC3 stimulates Wnt signaling by facilitating/stabilizing the interaction between Wnt and its receptor Frizzled (Fzd). Consistent with this hypothesis, we found that GPC3 is able to bind both Wnt and Fzd. The binding of GPC3 to Fzd is mediated by the GPC3 glycosaminoglycan chains and by the cysteine rich domain of Fzd.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2015-03-26T19:18:11Z No. of bitstreams: 1 Caroline Brandi_S Sales Identificação....pdf: 2182912 bytes, checksum: 259ec8612d135428b26583b6a9303dcc (MD5) / Approved for entry into archive by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2015-03-26T19:18:22Z (GMT) No. of bitstreams: 1 Caroline Brandi_S Sales Identificação....pdf: 2182912 bytes, checksum: 259ec8612d135428b26583b6a9303dcc (MD5) / Made available in DSpace on 2015-03-26T19:18:22Z (GMT). No. of bitstreams: 1 Caroline Brandi_S Sales Identificação....pdf: 2182912 bytes, checksum: 259ec8612d135428b26583b6a9303dcc (MD5) Previous issue date: 2015 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / INTRODUÇÃO: A Via Hedgehog (HH) está ativada em algumas neoplasias humanas, incluindo o Carcinoma Escamocelular de Boca (CEB), o qual corresponde a mais de 95% dos casos diagnosticados na cavidade bucal. Os glipicans (GPC) participam como reguladores desta cascata, atenuando (GPC1 e GPC3) ou regulando positivamente (GPC5) a via HH. OBJETIVO: O objetivo deste trabalho foi avaliar o perfil de expressão dos genes GPC1, 3 e 5, associando-os com genes da via HH (SHH, PTCH1 e SMO) e VEGFA, bem como caracterizar a imunoexpressão das proteínas GPC, em CEB. MATERIAL E MÉTODOS: Trinta e um casos de CEB foram submetidas a reações de qPCR para os genes SHH, PTCH1, SMO, VEGFA, GPC1, 3 e 5. O RNA total foi extraído utilizando uma coluna composta por membrana de silica (Rneasy Mini Kit). O DNA complementar foi obtido com auxílio da enzima Superscript Vilo™. As reações de qPCR foram conduzidas no aparelho ViiA™ 7 Real-Time PCR System utilizando o sistema Taqman, sendo a quantificação relativa avaliada pelo método comparativo de Cq (ΔΔCQ). Vinte e seis CEBs, 9 casos de margens tumorais (MAT) e 4 casos de mucosa bucal não neoplásica (MNN) foram submetidos à reação imuno-histoquímica para as proteínas GPC1, GPC3, GPC5, CD105 e MCM3 utilizando o sistema polimérico AdvanceTM ou LSABTM. As análises das proteínas GPC1, 3 e 5 foram realizadas de acordo com os parâmetros semi-quantitativos descritos por Gurgel et al. (2008). O número de células MCM3 positivas e de vasos/mm² (microdensidade vascular- MDV) foram avaliados em 5 campos, sendo a mediana de e intervalo de confiança utilizados para agrupar os CEBs em alto e baixo perfil proliferativo (AP e BP) e alta e baixa MDV, respectivamente. A análise estatística foi realizada utilizando GraphPad Prism versão 6.03. RESULTADOS: Transcritos do gene GPC1 (26; 83,87%); GPC3 (n=22; 70,97%) e GPC5 (n=15; 48,38%) foram observados em CEBs. SHH RNAm foi detectado em 5 CEBs (16,13%). A maioria dos CEBS apresentou expressão gênica de PTCH1 (n=25; 80.6%), SMO (n=26; 83,87%) e VEGFA (n=28; 90,32%). Correlação positiva forte e estatisticamente significante foi demonstrada para GPC5 e PTCH1 (rs=0,60; p=0,02) e entre PTCH1 e VEGFA (rs=0,69; p=0,0003). Imunomarcação citoplasmática e membranar de GPC1 foi observada principalmente em epitélio de MNN (n=4;100%) e MAT (n=9; 100%), enquanto que uma perda de imunomarcação desta proteína foi detectada no parênquima do CEB. A imunoexpressão da proteína GPC3 estava ausente em MNN (n= 4; 100%) e MAT (n=9; 100%). O GPC3 ocorreu na membrana e citoplasma de células do parênquima, observadas principalmente na periferia das ilhas tumorais, predominando o escore 3+ (n=5; 19.23%) entre os CEBs positivos (n=23; 88,46%). Ausência de imunomarcação de GPC5 foi observada em MNN (n=4; 0%) e apenas 2 espécimes de MAT (n=2; 22,22%) apresentaram baixa imunoexpressão, escore 1+. GPC5 citoplasmático em células tumorais positivas predominou o escore 1+ (n=5; 38.46%). Ao mesmo tempo, GPC5 foi detectado em estroma de 13 (50%) CEBs, especialmente em células endoteliais e semelhantes a fibroblastos. A expressão dos genes avaliados foi similar em tumores com AP e BP, assim como foi independente da MDV. CONCLUSÕES: A correlação entre os transcritos GPC5 e PTCH1, bem como a superexpressão das proteínas GPC5 e GPC3 e perda de imunopositividade de GPC1 são consistentes com a participação destas proteoglicanas como reguladoras da via HH em CEB. O perfil de expressão do gene e proteína GPC1 sugere que este glipican pode participa da biologia tumoral como uma proteína supressora tumoral, enquanto GPC3 e GPC5 participariam oncoproteínas. A presença de GPC5 em estroma tumoral (células endoteliais e fibroblastos) pode estar associada a regulação da via HH neste compartimento do microambiente tumoral. / INTRODUCTION: The Hedgehog pathway is activated in some human neoplasms, including Oral Squamous Cell Carcinoma (OSCC), which account for more than 95% of all oral cancers diagnosed. Glypicans are involved in the regulation of HH pathway through GPC3 e GPC1 downregulation or/and GPC5 upregulation. AIM: The aim of this study was to evaluate the expression profile of GPC1, 3 and 5 genes, correlating to HH and VEGFA gene, even as to characterize the immunoexpression of these proteins at OSCC. MATERIAL AND METHODS: A total of 31 cases of OSCC were assessed by qPCR for the SHH, PTCH1, SMO, VEGFA, GPC1, GPC3 and GPC5 genes. The total RNA were extracted using silica membrane column (Rneasy Mini Kit). Complementary DNA was obtained using of Superscript ™ Vilo enzyme. The qPCR reactions were performed in VIIA™ 7 Real-Time PCR System using the Taqman enzime, and relative quantification (RQ) was evaluated by the comparative method of Cq (ΔΔCQ). Immunohistochemical reactions for GPC1, GPC3, GPC5, MCM3 and CD105 proteins was performed on twenty-six OSCC, 9 cases of tumor margins (TM) and 4 cases of non-neoplastic oral mucosa (NNM) using AdvanceTM or LSABTM system. The analysis of GPC1, 3 and 5 proteins were conducted according to the semi-quantitative parameters described by Gurgel et al. (2008). The number of MCM3 positive cells and vessels//mm² (microvessel density -MVD) were evaluated in 5-matching areas, and the median and confidence interval being used to group the OSCC in high and low proliferative profile (HP and LP) and high and low MDV, respectively. Statistical analysis were carried out with GraphPad Prism v.6.03. RESULTS: Transcripts of GPC1 (26; 83.87%), GPC3 (n=22; 70.97%) and GPC5 (n=15; 48.38%) genes were observed in OSCC. SHH mRNA was detected in 5 OSCC (16:13%), PTCH1 gene in 25 CEBs (80.6%), SMO in 26 (83.87%) and VEGFA in 28 (90.32%). Strong and statistically significant positive correlation was demonstrated for GPC5 and PTCH1 genes (rs=0.60; p= 0.02) and PTCH1 and VEGFA transcripts (rs = 0.69; p = 0.0003). Cytoplasmic and membrane immunostaining of GPC1 was mainly observed in epithelial MNN (n = 5; 100%) and MAT (n=9; 100%), while a reduction of this protein was detected in parenchymal cells. GPC3 protein were absent in MNN (n = 4; 0%) and MAT (n=9; 0%). The GPC3 occurred in the membrane and cytoplasm of parenchymal cells, mainly observed in the periphery of the tumor islands and the 3+ score was predominat (n=3; 11:56%) in positive OSCC. GPC5 positive tumor cells occurred in the cytoplasm, scored 1+ (n = 5; 38.46%). In addition, GPC5 was detected in the stroma of 13 (50%) OSCC, especially in endothelial and fibroblast cells. The gene expression was similar in tumors with HP and LP, and was independent of MDV. CONCLUSIONS: The correlation between the GPC5 and PTCH1 transcripts, as well the overexpression of GPC5 and GPC3 protein and the loss of GPC1 positive cells are consistent with the participation of these proteoglycans as regulators of HH pathway in OSCC. The gene and protein expression profile of GPC3 indicate that this proteins participates in tumor biology as a tumor suppressor protein, while GPC5 and GPC3 function as oncoproteins. The presence of GPC5 in tumor stroma (endothelial cells and fibroblasts) could be associated with the regulation of the HH pathway in this compartment of the tumor microenvironment.

In der vorliegenden Arbeit konnte mit der PC12 Zellinie und dem MTT Viabilitätsassay Glypikan, ein Glykosylphosphatidylinositol-verankertes Heparan Sulfat, als Rezeptor identifiziert werden, der die toxischen Effekte von [beta]-Amyloid vermittelt. Kompetitive Substanzen im Medium, enzymatische Behandlung der Zelloberfläche und Block spezifischer zellulärer Synthesewege wurden eingesetzt. [beta]-Amyloid wird eine zentrale Rolle in der Pathogenese der Alzheimer'schen Krankheit zugeschrieben. Eine Intervention auf Ebene der [beta]-Amyloid-Rezeptor Interaktion könnte einen Therapieansatz für die Alzheimer'sche Krankheit darstellen. Weitere Experimente sind notwendig, um die Rolle von Glypikan bei der Vermittlung der [beta]-Amyloid Toxizität im Alzheimer-Gehirn zu untersuchen, z.B. in der Primärkultur von Neuronen und Mikroglia oder im lebenden Hirnschnitt. Eine ähnliche anatomische Verteilung der Glypikanexpression im Rattenhirn und der neurofibrillären Bündel im Alzheimergehirn wurde gefunden. Eine mögliche Rolle für Glypikan bei der Entstehung der neurofibrillären Bündel muß im Autopsiegehirn von Alzheimerpatienten überprüft werden. / [beta]-amyloid is thought to play a major role in the pathogenesis of Alzheimer's disease. In PC12 cell culture, competitive substances in cell culture medium, enzymatic cell surface treatment und block of specific cell synthesis pathways were used to identify glypican, a GPI- anchored heparan sulfate proteoglycan, as a receptor that mediates the toxic effects of [beta]-amyloid.

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La possibilité de programmer une cellule dans le but de produire une protéine d’intérêt est apparue au début des années 1970 avec l’essor du génie génétique. Environ dix années plus tard, l’insuline issue de la plateforme de production microbienne Escherichia coli, fut la première protéine recombinante (r-protéine) humaine commercialisée. Les défis associés à la production de r-protéines plus complexes et glycosylées ont amené l’industrie biopharmaceutique à développer des systèmes d’expression en cellules de mammifères. Ces derniers permettent d’obtenir des protéines humaines correctement repliées et de ce fait, biologiquement actives. Afin de transférer le gène d’intérêt dans les cellules de mammifères, le polyéthylènimine (PEI) est certainement un des vecteurs synthétiques le plus utilisé en raison de son efficacité, mais aussi sa simplicité d’élaboration, son faible coût et sa stabilité en solution qui facilite son utilisation. Il est donc largement employé dans le contexte de la production de r-protéines à grande échelle et fait l’objet d’intenses recherches dans le domaine de la thérapie génique non virale. Le PEI est capable de condenser efficacement l’ADN plasmidique (vecteur d’expression contenant le gène d’intérêt) pour former des complexes de petites tailles appelés polyplexes. Ces derniers doivent contourner plusieurs étapes limitantes afin de délivrer le gène d’intérêt au noyau de la cellule hôte. Dans les conditions optimales du transfert de gène par le PEI, les polyplexes arborent une charge positive nette interagissant de manière électrostatique avec les protéoglycanes à héparane sulfate (HSPG) qui décorent la surface cellulaire. On observe deux familles d’HSPG exprimés en abondance à la surface des cellules de mammifères : les syndécanes (4 membres, SDC1-4) et les glypicanes (6 membres, GPC1-6). Si l’implication des HSPG dans l’attachement cellulaire des polyplexes est aujourd’hui largement acceptée, leur rôle individuel vis-à-vis de cet attachement et des étapes subséquentes du transfert de gène reste à confirmer. Après avoir optimisées les conditions de transfection des cellules de mammifères CHO et HEK293 dans le but de produire des r-protéines secrétées, nous avons entrepris des cinétiques de capture, d’internalisation des polyplexes et aussi d’expression du transgène afin de mieux comprendre le processus de transfert de gène. Nous avons pu observer des différences au niveau de ces paramètres de transfection dépendamment du système d’expression et des caractéristiques structurelles du PEI utilisé. Ces résultats présentés sous forme d’articles scientifiques constituent une base solide de l’enchaînement dans le temps des évènements essentiels à une transfection efficace des cellules CHO et HEK293 par le PEI. Chaque type cellulaire possède un profil d’expression des HSPG qui lui est propre, ces derniers étant plus ou moins permissifs au transfert de gène. En effet, une étude menée dans notre laboratoire montre que les SDC1 et SDC2 ont des rôles opposés vis-à-vis du transfert de gène. Alors que tous deux sont capables de lier les polyplexes, l’expression de SDC1 permet leur internalisation contrairement à l’expression de SDC2 qui l’inhibe. De plus, lorsque le SDC1 est exprimé à la surface des cellules HEK293, l’efficacité de transfection est augmentée de douze pourcents. En utilisant la capacité de SDC1 à induire l’internalisation des polyplexes, nous avons étudié le trafic intracellulaire des complexes SDC1 / polyplexes dans les cellules HEK293. De plus, nos observations suggèrent une nouvelle voie par laquelle les polyplexes pourraient atteindre efficacement le noyau cellulaire. Dans le contexte du transfert de gène, les HSPG sont essentiellement étudiés dans leur globalité. S’il est vrai que le rôle des syndécanes dans ce contexte est le sujet de quelques études, celui des glypicanes est inexploré. Grâce à une série de traitements chimiques et enzymatiques visant une approche « perte de fonction », l’importance de la sulfatation comme modification post-traductionnelle, l’effet des chaînes d’héparanes sulfates mais aussi des glypicanes sur l’attachement, l’internalisation des polyplexes, et l’expression du transgène ont été étudiés dans les cellules CHO et HEK293. L’ensemble de nos observations indique clairement que le rôle des HSPG dans le transfert de gène devrait être investigué individuellement plutôt que collectivement. En effet, le rôle spécifique de chaque membre des HSPG sur la capture des polyplexes et leur permissivité à l’expression génique demeure encore inconnu. En exprimant de manière transitoire chaque membre des syndécanes et glypicanes à la surface des cellules CHO, nous avons déterminé leur effet inhibiteur ou activateur sur la capture des polyplexes sans pouvoir conclure quant à l’effet de cette surexpression sur l’efficacité de transfection. Par contre, lorsqu’ils sont présents dans le milieu de culture, le domaine extracellulaire des HSPG réduit l’efficacité de transfection des cellules CHO sans induire la dissociation des polyplexes. Curieusement, lorsque chaque HSPG est exprimé de manière stable dans les cellules CHO, seulement une légère modulation de l’expression du transgène a pu être observée. Ces travaux ont contribué à la compréhension des mécanismes d'action du vecteur polycationique polyéthylènimine et à préciser le rôle des protéoglycanes à héparane sulfate dans le transfert de gène des cellules CHO et HEK293. / With the aim to express a protein of interest, the transfer of exogenous genetic material into host cells was established in early 70s with the development of genetic engineering. Approximately ten years later, insulin was the first human recombinant protein (r-protein) produced at large scale in Escherichia coli and commercialized. Challenges associated with the production of more complex and glycosylated r-proteins brought the pharmaceutical industry to develop mammalian expression platforms. Thus, the expressed r-proteins are correctly folded and biologically actives. As a means to transfer genetic materials of interest into mammalian cells, the synthetic vector polyethylenimine (PEI) is probably the most popular due to its efficacy, ease of use, cost-effectiveness and stability in solution. Consequently, PEI is largely employed for the production of r-proteins by large scale and extensively studied in the context of non-viral gene therapy. PEI is capable to efficiently condense plasmid DNA (expression vector containing the gene of interest) to form small nanoparticles termed polyplexes. The latter must circumvent several steps to deliver the gene of interest to the cell nucleus. When formed at the optimum conditions, polyplexes exhibit a net positive charge which can interact electrostatically with negatively charged heparan sulfate proteoglycans (HSPG) located at the cell surface. There are two major families of HSPG that are largely expressed at the surface of mammalian cells: the syndecans (4 members, SDC1-4) and the glypicans (6 members, GPC1-6). Although it is generally accepted that HSPG are involved in the binding of polyplexes, their individual role toward polyplex binding and the subsequent phases of gene transfer need to be confirmed. Following optimization of the mammalian CHO and HEK293 cells transfection conditions, we undertook an in-depth study of polyplexes uptake, internalization kinetics, as well as transgene expression kinetics with the aim to better understand the mechanisms underlying gene transfer. We observed several contrasting differences between the two cell lines and the type of PEI used. Our results presented as a scientific article, establish strong basis of the gene transfer process over-time. Every cell type possesses its own expression profile of HSPG which can display individual potency toward gene transfer. Indeed, a preliminary study conducted in our laboratory showed that SDC1 and SDC2 have distinct features with regard to gene transfer. While both are capable to bind polyplexes at the cell surface, the expression of SDC1 enhances polyplexes internalization whereas the expression of SDC2 drastically inhibits it. Furthermore, when SDC1 is expressed at the surface of HEK293 cells, the transfection efficiency is increased by twelve percent compared to control cells. By using the ability of SDC1 to mediate efficient internalization of polyplexes, we have studied the intracellular traffic of SDC1 / polyplexes complexes. Our conclusions lead to new insights concerning the path by which polyplexes can mediate efficient transfection. In the context of gene transfer, HSPG have been essentially studied in their entirety. Although the role of syndecans is the subject of some studies, that of glypicans is unexplored. Thanks to a series of chemical and enzymatic treatments leading to « loss of functions », the importance of sulfation as post-translational modification, the effect of HS chains and of glypicans on the attachment, internalization of polyplexes as well as transgene expression were investigated in CHO and HEK293 cells. Taken together, our observations indicate clearly that the role of HSPG should be investigated individually instead of collectively. Consequently, the individual potency of each HSPG member regarding gene transfer remains to be defined. We demonstrated that, in fact, the transient expression of some HSPG in CHO cells have a beneficial effect on polyplexes uptake while others have a negative effect. Unfortunately, this method did not allow concluding about their effect on transfection efficacy. However, when present in the culture medium, the extracellular domain of HSPG decreases transfection efficacy of CHO cells without inducing polyplexes dissociation. Strangely, when each HSPG is stably expressed in CHO cells, only subtle modulations of the gene expression level were observed. This study contributed to a better understanding of the mechanisms underlying PEI mediated gene transfer in CHO and HEK293 cells and clarify the role of HSPG in gene transfer.