Human umbilical endothelial cells (HUVECs) have a sex: characterisation of the phenotype of male and female cells

Background

Human umbilical endothelial cells (HUVECs) are widely used to study the endothelial physiology and pathology that might be involved in sex and gender differences detected at the cardiovascular level. This study evaluated whether HUVECs are sexually dimorphic in their morphological, proliferative and migratory properties and in the gene and protein expression of oestrogen and androgen receptors and nitric oxide synthase 3 (NOS3). Moreover, because autophagy is influenced by sex, its degree was analysed in male and female HUVECs (MHUVECs and FHUVECs).

Methods

Umbilical cords from healthy, normal weight male and female neonates born to healthy non-obese and non-smoking women were studied. HUVEC morphology was analysed by electron microscopy, and their function was investigated by proliferation, viability, wound healing and chemotaxis assays. Gene and protein expression for oestrogen and androgen receptors and for NOS3 were evaluated by real-time PCR and Western blotting, respectively, and the expression of the primary molecules involved in autophagy regulation [protein kinase B (Akt), mammalian target of rapamycin (mTOR), beclin-1 and microtubule-associated protein 1 light chain 3 (LC3)] were detected by Western blotting.

Results

Cell proliferation, migration NOS3 mRNA and protein expression were significantly higher in FHUVECs than in MHUVECs. Conversely, beclin-1 and the LC3-II/LC3-I ratio were higher in MHUVECs than in FHUVECs, indicating that male cells are more autophagic than female cells. The expression of oestrogen and androgen receptor genes and proteins, the protein expression of Akt and mTOR and cellular size and shape were not influenced by sex. Body weights of male and female neonates were not significantly different, but the weight of male babies positively correlated with the weight of the mother, suggesting that the mother’s weight may exert a different influence on male and female babies.

Conclusions

The results indicate that sex differences exist in prenatal life and are parameter-specific, suggesting that HUVECs of both sexes should be used as an in vitro model to increase the quality and the translational value of research. The sex differences observed in HUVECs could be relevant in explaining the diseases of adulthood because endothelial dysfunction has a crucial role in the pathogenesis of cardiovascular diseases, diabetes mellitus, neurodegeneration and immune disease.

     

Co-localization of susceptibility loci for psoriasis (PSORS4) and atopic dermatitis (ATOD2) on human chromosome 1q21

Psoriasis (PS) is a chronic inflammatory skin disorder characterized by keratinocyte hyperproliferation and altered differentiation. Atopic dermatitis (ATOD) is a chronic inflammatory, pruritic and eczematous disease frequently associated with respiratory atopy. These diseases are associated with distinct immunologic abnormalities and represent typical examples of complex diseases triggered by both genetic and environmental factors, as demonstrated by independent twin studies. Genome wide linkage studies have mapped susceptibility loci on several chromosomes (PSORS1-9; ATOD1-5). Four of them overlap on chromosomes 1q21, 3q21, 17q25 and 20p although ATOD is quite distinct from PS and these two diseases rarely occur together in the same patient. An association fine-mapping study has been performed to refine PSORS4 and ATOD2 susceptibility loci on chromosome 1q21 analyzing two independently collected cohorts of 128 PS and 120 ATOD trios. Genotype and haplotype analysis of PSORS4 and ATOD2 led us to detect significant p value for haplotypes defined by MIDDLE and ENDAL16 markers in both PS (p = 0.0000036) and ATOD (p = 0.0276), suggesting a strict co-localization within an interval of 42 kb. This genomic interval contains a single gene, LOR, encoding for loricrin. Polymorphic markers mapping in regulatory and coding regions did not show evidence of association in neither of the two diseases. However, expression profiles of LOR in skin biopsies have shown reduced levels in PS and increased levels in ATOD, suggesting the existence of a specific misregulation in LOR mRNA production.     

An unconventional road for the secretion of transglutaminase in pollen tubes?

The transglutaminase (TGase) is present in the pollen tube where it most likely participates in the regulation of different activities including the organization of cytoskeletal elements (microtubules and actin filaments). In addition to a cytosolic form of TGase, new data suggest the existence of TGase forms associated with the internal membranes and with the cell wall of pollen tubes. This different localization extends the functional range of pollen TGase but also raises the question how TGase can be precisely (and in harmony with the pollen tube growth) redistributed in different cellular compartments. The discovery that TGase exists as different isoforms may suggest a pathway to achieve this result.     

Fluorescent ligands for adenosine receptors

Interest is increasing in developing fluorescent ligands for characterization of adenosine receptors (ARs), which hold a promise of usefulness in the drug discovery process. The size of a strategically labeled AR ligand can be greatly increased after the attachment of a fluorophore. The choice of dye moiety (e.g. Alexa Fluor 488), attachment point and linker length can alter the selectivity and potency of the parent molecule. Fluorescent derivatives of adenosine agonists and antagonists (e.g. XAC and other heterocyclic antagonist scaffolds) have been synthesized and characterized pharmacologically. Some are useful AR probes for flow cytometry, fluorescence correlation spectroscopy, fluorescence microscopy, fluorescence polarization, fluorescence resonance energy transfer, and scanning confocal microscopy. Thus, the approach of fluorescent labeled GPCR ligands, including those for ARs, is a growing dynamic research field.     

Regulatory effect of IFN-kappa,a novel type I IFN,on cytokine production by cells of the innate immune system

IFN-kappa is a recently identified type I IFN that exhibits both structural and functional homology with the other type I IFN subclasses. In this study, we have investigated the effect of IFN-kappa on cells of the innate immune system by comparing cytokine release following treatment of human cells with either IFN-kappa or two recombinant IFN subtypes, IFN-beta and IFN-alpha2a. Although IFN-alpha2a failed to stimulate monocyte cytokine secretion, IFN-kappa, like IFN-beta, induced the release of several cytokines from both monocytes and dendritic cells, without the requirement of a costimulatory signal. IFN-kappa was particularly effective in inhibiting inducible IL-12 release from monocytes. Unlike IFN-beta, IFN-kappa did not induce release of IFN-gamma by PBL. Expression of the IFN-kappa mRNA was observed in resting dendritic cells and monocytes, and it was up-regulated by IFN-gamma stimulation in monocytes, while IFN-beta mRNA was minimally detectable under the same conditions. Monocyte and dendritic cell expression of IFN-kappa was also confirmed in vivo in chronic lesions of psoriasis vulgaris and atopic dermatitis. Finally, biosensor-based binding kinetic analysis revealed that IFN-kappa, like IFN-beta, binds strongly to heparin (K(d): 2.1 nM), suggesting that the cytokine can be retained close to the local site of production. The pattern of cytokines induced by IFN-kappa in monocytes, coupled with the unique induction of IFN-kappa mRNA by IFN-gamma, indicates a potential role for IFN-kappa in the regulation of immune cell functions.     

A comparative study on axial coordination and ligand binding in ferric mini myoglobin and horse heart myoglobin

The absorption and resonance Raman spectra and the azide binding kinetics of ferric horse heart myoglobin (Mb) and mini myoglobin (a chemically truncated form of horse heart Mb containing residues 32-139) have been compared. The steady-state spectra show that an additional six-coordinated low-spin form (not present in entire horse heart Mb, which is purely six-coordinated high spin) predominates in mini Mb. The distal histidine is possibly the sixth ligand in this species. The presence of two species corresponds to a kinetic biphasicity for mini Mb that is not observed for horse heart Mb. Azide binds to horse heart Mb much more slowly than to sperm whale Mb. This difference may result from a sterically hindered distal pocket in horse heart Mb. In both cases, the rate constants level off at high azide concentrations, implying the existence of a rate-limiting step (likely referable to the dissociation of the axial sixth ligand). The faster rate constant of mini Mb is similar to that of sperm whale Mb, whereas the slower one is similar to that of entire horse heart Mb.     

Effect of thienyl groups on the photoisomerization and rotamerism of symmetric and asymmetric diaryl-ethenes and diaryl-butadienes.

Five symmetric (bis-substituted) and asymmetric (mono-substituted) analogues of E-stilbene and EE-1,4-diphenylbutadiene, where one or both the side aryls are 2'-thienyl or 3'-thienyl groups, have been studied by stationary and pulsed fluorimetric techniques, laser flash photolysis, conventional photochemical methods and theoretical calculations. The results obtained for these compounds and the comparison with those previously reported for three other compounds of the same series, allowed the effects of the position of the heteroatom and of the extension of the olefin chain on the excited state relaxation properties to be understood. The presence of one or two thienyl groups and their positional isomerism affect the spectral behaviour, the relaxation properties (radiative/reactive competition), the photoisomerization mechanism (singlet/triplet) and the ground state rotamerism. For the dienes containing the 3'-thienyl substituent(s), two rotamers were evidenced whose radiative and photochemical properties were obtained by selective excitation.     

Presence of specific prolactin binding sites in the rabbit hypothalamus

The presence of specific prolactin binding sites in membranes obtained from rabbit brain was investigated. Among the different brain areas studied (hypothalamus, cerebral cortex, cerebellum, olfactory bulbi and ponsmedulla) only the hypothalamus showed an evident specific binding (5.34% in male and 6.64% in female rabbits), whereas in the other brain regions the binding was lower or negligible. Scatchard analysis of the binding revealed the presence of high-affinity saturable binding sites. The study on hormonal specificity showed that the binding in the hypothalamus was inhibited by ovine and rat prolactin and by human GH, but not by many other polypeptide hormones.