In vitro impact of physiological shear stress on endothelial cells gene expression profile.

In the vascular system, the shear applied to the vascular wall activates mechano-sensors located on endothelial cells (ECs) leading to a modification in the gene expression profile. We applied laminar shear stress at 1 Pa on ECs for 6 h and measured by quantitative real time PCR the expression modulation of genes implied in inflammation (ICAM-1 and E-selectin), oxidative stress sensing (HO-1) and vascular tone modulation (eNOS). We showed that all these genes are shear stress inducible. ICAM-1 is more up-regulated than E-selectin suggesting different levels of implication in inflammatory responses and different modes of induction (SSRE, cytokine). Laminar shear stress induces an oxidative stress translated into HO-1 up-regulation, and a possible vasodilatation through the induction of eNOS. Our laminar shear stress system opens a novel and interesting frame in the evaluation of the impact on ECs and blood cells of new pharmacological substances injected in the bloodstream.

Initial adhesion of endothelial cells on polyelectrolyte multilayer films.

Polyelectrolyte multilayer films were recently investigated to favour attachment of Human Vein Umbilical Endothelial Cells (HUVECs) on non-adhesive surfaces. In this study, we evaluated the initial adhesion of HUVECs after 3 h of seeding on two polyelectrolyte multilayer films ending by poly(D-lysine) (PDL) or poly(allylamine hydrochloride) (PAH). In order to obtain information about initial adhesion of HUVECs, cell morphology as well as the expression of beta1 integrins, specific receptors of adhesion, were evaluated after 3 h of seeding on polyelectrolyte multilayer films. The data were also compared to PDL or PAH monolayers (polyelectrolytes terminating the multilayer architecture). The expression of beta1 integrins was not different, whatever are the studied surfaces. However, HUVECs spreading on polyelectrolyte multilayer films, in particular on PAH ending film, was more important as compared to polyelectrolyte monolayers or glass. In conclusion, the best initial adhesion conditions of HUVECs on polyelectrolyte films could not be elucidated, moreover the results suggested also that beta1 integrins could only play a limited role.

Decellularized umbilical artery treated with thin polyelectrolyte multilayer films: potential use in vascular engineering.

Decellularized allograft tissues have been identified as a potential extracellular matrix scaffold for tissue-engineered vascular substitutes. In order to improve the thromboresistance, it is necessary to pre-coat the intra-luminal vessel surface. Recently a new surface modification technique appeared, based on the alternate adsorption of positive and negative charged polyelectrolytes. Our objective was to develop an alternative vascular scaffold made of decellularized human umbilical arteries treated with a PAH/PSS polyelectrolyte multilayered film. The vessels luminal surfaces covered with the multilayer film were observed by electronic scanning microscopy. Our observations showed that the luminal surface is completely devoid of ECs following treatment with trypsin. A top view of the coated artery indicated that the multilayer uniformly covered internal surface of the vessels. The successful of the multilayer correct deposition and retention on the arterial wall were controlled by confocal microscopy using a fluorescent polyelectrolyte (rhodamine-PAH). The data suggest that decellularized cryopreserved arteries represent a potential scaffold for further vascular tissue engineering efforts. Moreover, the multilayer films can be used to coat biological surfaces and following the terminated layer (PAH or PSS), favour the cell adhesion or cell resistance.

Behaviour of endothelial cells seeded on thin polyelectrolyte multilayered films: a new biological scaffold.

The surface modification using thin polyelectrolyte multilayered films was proposed as a new scaffold material for different cell lines. In this study, we evaluated the possible use of polyelectrolyte multilayers as surface modification for the development of endothelial cells. In order to control the behaviour of endothelial cells, cell viability by MTT assay was studied. Moreover, the endothelial cell phenotype was checked and the expression of a leukocyte adhesion molecule (ICAM-1) was quantified. The behaviour of the cells on two polyelectrolyte multilayers was compared to cells on polystyrene, and two polyelectrolyte monolayers (terminating the multilayer architectures). The results have shown a better cell viability on the polyelectrolyte multilayers, inducing a higher cell number compared to polyelectrolyte monolayers after 1 and 3 days of culture. Moreover, the cells showed a normal morphology of cytoskeleton. The phenotype of the endothelial cells was kept and a low level of leukocyte adhesion molecules was observed. In conclusion, the polyelectrolyte multilayers can be considered as a potential surface modification procedure to enhance the development of endothelial cells on hydrophobic substrate and which can be applied to vascular tissue engineering.

Endothelial cells grown on thin polyelectrolyte mutlilayered films: an evaluation of a new versatile surface modification.

Endothelial cell seeding constitutes an appreciated method to improve blood compatibility of small-diameter vascular grafts. In this study, we report the development of a simple innovative technique based on multilayered polyelectrolyte films as cell adhesive substrates. Polyelectrolyte multilayered films ending by poly(sodium-4-styrenesulfonate)/poly(allylamine hydrochloride) (PSS/PAH) or poly(L-glutamic acid)/poly(D-lysine) (PGA/PDL) could enhance cell adhesion by modification of the physico-chemical properties of the surface. The biological responses of human umbilical vein endothelial cells seeded on the polyelectrolyte multilayer films, on PDL or PAH monolayers, and on control surfaces, were evaluated in terms of initial attachment, growth, cellular metabolic activity, endothelial phenotype, and adhesion. The results showed that polyelectrolyte multilayers neither induce cytotoxic effects nor alter the phenotype of the endothelial cells. The polyelectrolyte multilayered films enhanced initial cell attachment as compared to the polyelectrolyte monolayer. Cell growth observed on the films was similar to that on TCPS. Among the different coating tested, the film ending by PSS/PAH exhibited an excellent cellular biocompatibility and appeared to be the most interesting surface in terms of cellular adhesion and growth. Such films could be used to cover hydrophobic (cell resistant) substrates in order to promote cell colonization, thereby constituting an excellent material for endothelial cell seeding.

From hemorheology to vascular mechanobiology: An overview.

Almost all of the cells of the human body are subjected to mechanical stresses. In endothelial cells, mechanical stresses can vary from some milli-Pascal (shear stress) to one ore more Pascal (hydrostatic pressure). Now it is know that mechanical stresses have a decisive part cellular physiology. However, if the main biological effects of mechanical stress are well related, the mechanisms allowed the relation between mechanical stress to physiological phenomenon remain nearly unknown (mechanotransduction phenomenon). In this work, through personal results and published works, the authors considers all the effects of mechanical stresses and the possible hypothesis.

Human umbilical vein endothelial cells increase ex vivo expansion of human CD34(+) PBPC through IL-6 secretion.

BACKGROUND: Ex vivo expansion of hematopoietic stem cells (HSC) can help reduce cytopenia following transplantation, especially in NHL patients whose BM is deficient because of extensive chemotherapy. We have previously reported that human umbilical vein endothelial cells (HUVEC) can contribute to improved PBPC expansion when used in co-culture with CD34(+) cells. METHODS: We evaluated the roles of direct HUVEC CD34(+) contact and HUVEC-produced soluble factors. We cultured CD34(+) PBPC harvested from NHL patients in four different conditions: (1) liquid culture without HUVEC; (2) co-culture in contact with HUVEC; (3) co-culture with HUVEC but without direct contact; (4) liquid culture with HUVEC-conditioned medium (CM). Thrombopoietin (Tpo), Flk2Flt3 ligand (FL) and c-kit ligand (KL) with or without rhIL-6 were added to these four culture conditions. RESULTS AND DISCUSSION: Our results showed that HUVEC co-culture or addition of HUVEC-CM to Tpo, FL and KL (TFK) improved CD34(+) PBPC expansion compared with liquid culture, as determined by total viable nucleated cells (TNC), colony-forming cell assay (CFC) and week-6 cobblestone area-forming cells (Wk-6 CAFC) expansions. Non-contact culture led to similar PBPC expansion as contact co-culture; moreover, HUVEC-CM improved PBPC expansion. However, when rhIL-6 was added to HUVEC-CM with TFK, no significant difference was observed. Finally, high quantities of IL-6 were detected in HUVEC-CM and addition of anti-IL-6 Ab inhibited the positive effect of HUVEC on PBPC expansion. Our results thus suggest that HUVEC may improve PBPC expansion, at least through IL-6 secretion.

Colocalization of delta sleep inducing peptide and luteinizing hormone releasing hormone in neurosecretory vesicles in rat median eminence.

The colocalization of immunoreactivities similar to delta sleep inducing peptide (DSIP) and luteinizing hormone releasing hormone (LH-RH) was investigated by light and electron microscopic immunocytochemistry in the rat median eminence. At the light microscopic level, DSIP and LH-RH immunostained fibers, and varicosities exhibited a similar distribution pattern throughout the median eminence. Immunoreactive axons were mainly found in the lateral part of the external layer. Using an elution-restaining technique, the coexistence of LH-RH and DSIP immunoreactivities was observed in most labelled axons. To determine the intracellular localization of DSIP and LH-RH, we used double immunocytochemical labelling with species-specific antibodies and secondary antibodies conjugated to different sizes of gold particles. The two peptides were found colocalized in single axons. Immunoreactive terminals frequently showed direct membrane apposition with tanycyte processes but rare contacts with portal capillaries. No staining was observed in tanycytes and ependymal or glial elements. Moreover, we could demonstrate that LH-RH and DSIP (or a closely related molecular form) are contained not only in the same axons, but also in the same approximately 100-nm dense-core vesicles, suggesting cosecretion of these peptides.