Tamara Hermosilla

DIRECT THY-1/αvβ3 INTEGRIN INTERACTION MEDIATES NEURON TO ASTROCYTE COMMUNICATION

Thy-1 is an abundant neuronal glycoprotein of poorly defined function. We recently provided evidence indicating that Thy-1 clusters a beta3-containing integrin in astrocytes to induce tyrosine phosphorylation, RhoA activation and the formation of focal adhesions and stress fibers. To date, the alpha subunit partner of beta3 integrin in DI TNC1 astrocytes is unknown. Similarly, the ability of neuronal, membrane-bound Thy-1 to trigger astrocyte signaling via integrin engagement remains speculation. Here, evidence that alphav forms an alphavbeta3 heterodimer in DI TNC1 astrocytes was obtained. In neuron-astrocyte association assays, the presence of either anti-alphav or anti-beta3 integrin antibodies reduced cell-cell interaction demonstrating the requirement of both integrin subunits for this association. Moreover, anti-Thy-1 antibodies blocked stimulation of astrocytes by neurons but not the binding of these two cell types. Thus, neuron-astrocyte association involved binding between molecular components in addition to the Thy-1-integrin; however, the signaling events leading to focal adhesion formation in astrocytes depended exclusively on the latter interaction. Additionally, wild-type (RLD) but not mutated (RLE) Thy-1 was shown to directly interact with alphavbeta3 integrin by Surface Plasmon Resonance analysis. This interaction was promoted by divalent cations and was species-independent. Together, these results demonstrate that the alphavbeta3 integrin heterodimer interacts directly with Thy-1 present on neuronal cells to stimulate astrocytes.

Ion channel formation by Alzheimer's disease amyloid β-peptide (Aβ40) in unilamellar liposomes is determined by anionic phospholipids

Abstract Incorporation of Alzheimers disease amyloid β-proteins (AβPs) across natural and artificial bilayer membranes leads to the formation of cation-selective channels. To study the peptide–membrane interactions involved in channel formation, we used cation reporter dyes to measure AβP-induced influx of Na + , Ca 2+ , and K + into liposomes formed from phosphatidylserine (PS), phosphatidylinositol (PI) and phosphatidylcholine (PC). We found that Aβ40, but not Aβ40-1 or Aβ28, caused a dose-dependent increase in the concentration of each cation in the lumen of liposomes formed from the acidic phospholipids PS and PI. The Aβ40-induced changes in cation concentration, which we attribute to ion entry through Aβ40 channels, were not observed when using liposomes formed from the neutral phospholipid PC. Using mixtures of phospholipids, the magnitude of the AβP40-induced ion entry increased with the acidic phospholipid content of the liposomes, with entry being observed with as little as 5% PS or PI. Thus, while negatively charged phospholipids are required for formation of cation-permeable channels by Aβ40, a small amount is sufficient to support the process. These results have implications for the mechanisms of AβP cytotoxicity, suggesting that even a small amount of externalized negative charge could render cells susceptible to the deleterious effects of unregulated ion influx through AβP channels.

Recognition of an immunogenetically selected Trypanosoma cruzi antigen by seropositive chagasic human sera

If the H-2 congenic mouse strains A.SW (H-2n) and A.CA (H-2f), are infected with Trypanosoma cruzi, a 45 kDa protein (Tc45), present in cultured epimastigotes and blood trypomastigotes, is recognized only by the A.SW strain sera. In order to explore the possibility that among seropositive humans the response to Tc45 is also highly variable, 81 chagasic human sera (as defined by the HemAve agglutination test, Polychaco S.A.I.C., Buenos Aires, Argentina) were tested in a direct (epimastigote antigenic complex directly bound to the solid phase) and indirect immunoradiometric assay (IRMA) (Tc45, from a partially purified preparation, bound to the solid phase, by means of a monoclonal antibody). Sixty nine of these sera reacted in both the direct and indirect assays, 11 were negative in both assays (these samples may correspond to false positives detected by the commercial agglutination test) and only one reacted with the antigenic complex but not with Tc45. Reactivity of the human sera with the epimastigote antigenic extract was relatively homogenous, while reactivity with Tc45 was extremely variable. No statistical correlation was determined between the two variables. Given the high variability of the human response to Tc45, ranging from negative to highly positive, together with the immunogenetic restriction previously described in the murine model, we speculate that human MHC may also modulate the response to this molecule.

Endotoxin Induces Fibrosis in Vascular Endothelial Cells through a Mechanism Dependent on Transient Receptor Protein Melastatin 7 Activity

The pathogenesis of systemic inflammatory diseases, including endotoxemia-derived sepsis syndrome, is characterized by endothelial dysfunction. It has been demonstrated that the endotoxin lipopolysaccharide (LPS) induces the conversion of endothelial cells (ECs) into activated fibroblasts through endothelialtomesenchymal transition mechanism. Fibrogenesis is highly dependent on intracellular Ca2+ concentration increases through the participation of calcium channels. However, the specific molecular identity of the calcium channel that mediates the Ca2+ influx during endotoxin-induced endothelial fibrosis is still unknown. Transient receptor potential melastatin 7 (TRPM7) is a calcium channel that is expressed in many cell types, including ECs. TRPM7 is involved in a number of crucial processes such as the conversion of fibroblasts into activated fibroblasts, or myofibroblasts, being responsible for the development of several characteristics of them. However, the role of the TRPM7 ion channel in endotoxin-induced endothelial fibrosis is unknown. Thus, our aim was to study whether the TRPM7 calcium channel participates in endotoxin-induced endothelial fibrosis. Using primary cultures of ECs, we demonstrated that TRPM7 is a crucial protein involved in endotoxin-induced endothelial fibrosis. Suppression of TRPM7 expression protected ECs from the fibrogenic process stimulated by endotoxin. Downregulation of TRPM7 prevented the endotoxin-induced endothelial markers decrease and fibrotic genes increase in ECs. In addition, TRPM7 downregulation abolished the endotoxin-induced increase in ECM proteins in ECs. Furthermore, we showed that intracellular Ca2+ levels were greatly increased upon LPS challenge in a mechanism dependent on TRPM7 expression. These results demonstrate that TRPM7 is a key protein involved in the mechanism underlying endotoxin-induced endothelial fibrosis.

Immunomodulation of LPS ability to induce the local Shwartzman reaction.

Immunologically, the septic shock is a natural model of immunomediated vascular pathology where the interaction between cytokines and the endothelium mediates the syndrome and lethality. Tumour necrosis factor (TNF), a non‐species‐specific cytokine, has outstanding pleiotropic activities as an important mediator of the septic shock syndrome. In rabbits, passive immunization with anti‐lipopolysaccharide (LPS) polyclonal antibodies prior to the intravenous (i.v.) injection of LPS inhibits the haemorrhagic necrotic lesion characteristic of the local Shwartzman reaction (an excellent localized in vivo correlate of the septic shock). Paradoxically, tested in an ex vivo assay (short‐term whole human blood culture, stimulated with LPS), these antibodies mediated an increase in TNF production by mononuclear phagocytes and, in the rabbit model, they induced an increase in body temperature, as compared with the pre‐immune reagent. Although anchoring of immune complexes containing LPS to receptors (Fc or C4b‐C3b) on circulating monocytes may facilitate the access of LPS to these cells, access to localized, LPS‐sensitized macrophages may be impaired. Consequently inhibition of the local Shwartzman reaction and increased TNF production in the ex vivo system were observed. Concordantly, the higher temperature in the passively immunized animals may be a consequence of a higher, immune complex‐induced, systemic TNF production. These experimental results suggest that the use of anti‐LPS immunoglobulins, as a potential immunotherapy for septic shock syndrome in vertebrates, may lead to increased TNF production, with adverse effects such as the pyrogenic.

Trypanosoma cruzi: H2 complex and genetic background influence on the humoral immune response against epimastigotes.

Using A.SW, A.CA, B10.S and B10.M congenic mouse strains, we measured the IgG specific humoral immune responses against sonicated and live Trypanosoma cruzi epimastigotes. Genes located in the A background (A.SW and A.CA strains) mediate higher IgG responses against the parasite antigenic complexes than those located in the B background (strains B10.S and B10.M), regardless of the H2 haplotypes. Thus, non H2 genetic elements seem to be more important in determining differences in the total IgG immune response against T. cruzi. Whether a detectable H2 effect, in favor of the H2(s) haplotype, occurred in the A or B background, was contingent on the immunisation protocol used. Thus, the H2(s) haplotype mediates a higher IgG response in the A background, if immunised with live epimastigotes, and in the B background against sonicated epimastigotes. Most likely this represents a complex sequence of events, controlled by non-MHC genes, involving antigen handling and processing and depending on the physical form of antigen delivery.

Cavβ2 transcription start site variants modulate calcium handling in newborn rat cardiomyocytes

In the heart, the main pathway for calcium influx is mediated by L-type calcium channels, a multi-subunit complex composed of the pore-forming subunit CaV1.2 and the auxiliary subunits CaVα2δ1 and CaVβ2. To date, five distinct CaVβ2 transcriptional start site (TSS) variants (CaVβ2a-e) varying only in the composition and length of the N-terminal domain have been described, each of them granting distinct biophysical properties to the L-type current. However, the physiological role of these variants in Ca2+ handling in the native tissue has not been explored. Our results show that four of these variants are present in neonatal rat cardiomyocytes. The contribution of those CaVβ2 TSS variants on endogenous L-type current and Ca2+ handling was explored by adenoviral-mediated overexpression of each CaVβ2 variant in cultured newborn rat cardiomyocytes. As expected, all CaVβ2 TSS variants increased L-type current density and produced distinctive changes on L-type calcium channel (LTCC) current activation and inactivation kinetics. The characteristics of the induced calcium transients were dependent on the TSS variant overexpressed. Moreover, the amplitude of the calcium transients varied depending on the subunit involved, being higher in cardiomyocytes transduced with CaVβ2a and smaller in CaVβ2d. Interestingly, the contribution of Ca2+ influx and Ca2+ release on total calcium transients, as well as the sarcoplasmic calcium content, was found to be TSS-variant-dependent. Remarkably, determination of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) messenger RNA (mRNA) abundance and cell size change indicates that CaVβ2 TSS variants modulate the cardiomyocyte hypertrophic state. In summary, we demonstrate that expression of individual CaVβ2 TSS variants regulates calcium handling in cardiomyocytes and, consequently, has significant repercussion in the development of hypertrophy.

Prolonged AT1R activation induces CaV1.2 channel internalization in rat cardiomyocytes

The cardiac L-type calcium channel is a multi-subunit complex that requires co-assembling of the pore-forming subunit CaV1.2 with auxiliary subunits CaVα2δ and CaVβ. Its traffic has been shown to be controlled by these subunits and by the activation of various G-protein coupled receptors (GPCR). Here, we explore the consequences of the prolonged activation of angiotensin receptor type 1 (AT1R) over CaV1.2 channel trafficking. Bioluminescence Resonance Energy Transfer (BRET) assay between β-arrestin and L-type channels in angiotensin II-stimulated cells was used to assess the functional consequence of AT1R activation, while immunofluorescence of adult rat cardiomyocytes revealed the effects of GPCR activation on CaV1.2 trafficking. Angiotensin II exposure results in β-arrestin1 recruitment to the channel complex and an apparent loss of CaV1.2 immunostaining at the T-tubules. Accordingly, angiotensin II stimulation causes a decrease in L-type current, Ca2+ transients and myocyte contractility, together with a faster repolarization phase of action potentials. Our results demonstrate that prolonged AT1R activation induces β-arrestin1 recruitment and the subsequent internalization of CaV1.2 channels with a half-dose of AngII on the order of 100 nM, suggesting that this effect depends on local renin-angiotensin system. This novel AT1R-dependent CaV1.2-trafficking modulation likely contributes to angiotensin II-mediated cardiac remodeling.