Optimising approaches to active immunotherapy of cancer

The design of therapeutic cancer vaccines is aimed at inducing high numbers and potent T cells that are able to target and eradicate malignant cells. This calls for close collaboration between cells of the innate immune system, in particular dendritic cells (DCs), and cells of the adaptive immune system, notably CD4+ helper T cells and CD8+ cytotoxic T cells. Therapeutic vaccines are aided by adjuvants, which can be, for example, Toll¬like Receptor agonists or agents promoting the cytosolic delivery of antigens, among others. Vaccination with long synthetic peptides (LSPs) is a promising strategy, as the requirement for their intracellular processing will mainly target LSPs to professional antigen presenting cells (APCs), hence avoiding the immune tolerance elicited by the presentation of antigens by non-professional APCs. The unique property of antigen cross-processing and cross-presentation activity by DCs plays an important role in eliciting antitumour immunity given that antigens from engulfed dead tumour cells require this distinct biological process to be processed and presented to CD8+T cells in the context of MHC class I molecules. DCs expressing the XCR1 chemokine receptor are characterised by their superior capability of antigen cross- presentation and priming of highly cytotoxic T lymphocyte (CTL) responses. Recently, XCR1 was found to be also expressed in tissue-residents DCs in humans, with a simitar transcriptional profile to that of cross- presenting murine DCs. This shed light into the value of harnessing this subtype of XCR1+ cross-presenting DCs for therapeutic vaccination of cancer. In this study, we explored ways of adjuvanting and optimising LSP therapeutic vaccinations by the use, in Part I, of the XCLl chemokine that selectively binds to the XCR1 receptor, as a mean to target antigen to the cross-presenting XCR1+ DCs; and in Part II, by the inclusion of Q.S21 in the LSP vaccine formulation, a saponin with adjuvant activity, as well as the ability to promote cytosolic delivery of LSP antigens due to its intrinsic cell membrane insertion activity. In Part I, we designed and produced XCLl-(OVA LSP)-Fc fusion proteins, and showed that their binding to XCR1+ DCs mediate their chemoattraction. In addition, therapeutic vaccinations adjuvanted with XCLl-(OVA LSP)-Fc fusion proteins significantly enhanced the OVA-specific CD8+ T cell response, and led to complete tumour regression in the EL4-OVA model, and significant control of tumour growth in the B16.0VA tumour model. With the aim to optimise the co-delivery of LSP antigen and XCLl to skin-draining lymph nodes we also tested immunisations using nanoparticle (NP)-conjugated OVA LSP in the presence or absence of XCLl chemokine. The NP-mediated delivery of LSP potentiated the CTL response seen in the blood of vaccinated mice, and NP-OVA LSP vaccine in the presence of XCLl led to higher blood frequencies of OVA-specific memory-precursor effector cells. Nevertheless, in these settings, the addition XCLl to NP-OVA LSP vaccine formulation did not increase its antitumour therapeutic effect. In the Part II, we assessed in HLA-A2/DR1 mice the immunogenicity of the Melan-AA27L LSP or the Melan-A26. 35 AA27l short synthetic peptide (SSP) used in conjunction with the saponin adjuvant QS21, aiming to identify a potent adjuvant formulation that elicits a quantitatively and qualitatively strong immune response to tumour antigens. We showed a high CTL immune response elicited by the use of Melan-A LSP or SSP with QS21, which both exerted similar killing capacity upon in vivo transfer of target cells expressing the Melan-A peptide in the context of HLA-A2 molecules. However, the response generated by the LSP immunisation comprised higher percentages of CD8+T cells of the central memory phenotype (CD44hl CD62L+ and CCR7+ CD62L+) than those of SSP immunisation, and most importantly, the strong LSP+QS21 response was strictly CD4+T cell-dependent, as shown upon CD4 T cell depletion. Altogether, these results suggest that both XCLl and QS21 may enhance the ability of LSP to prime CD8 specific T cell responses, and promote a long-term memory response. Therefore, these observations may have important implications for the design of protein or LSP-based cancer vaccines for specific immunotherapy of cancer -- Les vacans thérapeutiques contre le cancer visent à induire une forte et durable réponse immunitaire contre des cellules cancéreuses résiduelles. Cette réponse requiert la collaboration entre le système immunitaire inné, en particulier les cellules dendrites (DCs), et le système immunitaire adaptatif, en l'occurrence les lymphocytes TCD4 hdper et CD8 cytotoxiques. La mise au point d'adjuvants et de molécules mimant un agent pathogène tels les ligands TLRs ou d'autres agents facilitant l'internalisation d'antigènes, est essentielle pour casser la tolérance du système immunitaire contre les cellules cancéreuses afin de générer une réponse effectrice et mémoire contre la tumeur. L'utilisation de longs peptides synthétiques (LSPs) est une approche prometteuse du fait que leur présentation en tant qu'antigénes requiert leur internalisation et leur transformation par les cellules dendrites (DCs, qui sont les mieux à même d'éviter la tolérance immunitaire. Récemment une sous-population de DCs exprimant le récepteur XCR1 a été décrite comme ayant une capacité supérieure dans la cross-présentation d'antigènes, d'où un intérêt à développer des vaccins ciblant les DCs exprimant le XCR1. Durant ma thèse de doctorat, j'ai exploré différentes approches pour optimiser les vaccins avec LSPs. La première partie visait à cibler les XCR1-DCs à l'aide de la chemokine XCL1 spécifique du récepteur XCR1, soit sou s la forme de protéine de fusion XCL1-OVA LSP-Fc, soit associée à des nanoparticules. La deuxième partie a consisté à tester l'association des LSPs avec I adjuvant QS21 dérivant d'une saponine dans le but d'optimiser l'internalisation cytosolique des longs peptides. Les protéines de fusion XCLl-OVA-Fc développées dans la première partie de mon travail, ont démontré leur capacité de liaison spécifique sur les XCRl-DCs associée à leur capacité de chemo-attractio. Lorsque inclues dans une mmunisation de souris porteuse de tumeurs établies, ces protéines de fusion XCL1-0VA LSP-Fc et XCLl-Fc plus OVA LSP ont induites une forte réponse CDS OVA spécifique permettant la complète régression des tumeurs de modèle EL4- 0VA et un retard de croissance significatif de tumeurs de type B16-0VA. Dans le but d'optimiser le drainage des LSPs vers es noyaux lymphatiques, nous avons également testé les LSPs fixés de manière covalente à des nanoparticules co- injectees ou non avec la chemokine XCL1. Cette formulation a également permis une forte réponse CD8 accompagnée d'un effet thérapeutique significatif, mais l'addition de la chemokine XCL1 n'a pas ajouté d'effet anti-tumeur supplémentaire. Dans la deuxième partie de ma thèse, j'ai comparé l'immunogénicité de l'antigène humain Melan A soit sous la forme d un LSP incluant un épitope CD4 et CD8 ou sous la forme d'un peptide ne contenant que l'épitope CD8 (SSP) Les peptides ont été formulés avec l'adjuvant QS21 et testés dans un modèle de souris transgéniques pour les MHC let II humains, respectivement le HLA-A2 et DR1. Les deux peptides LSP et SSP ont généré une forte réponse CD8 similaire assoc.ee a une capacité cytotoxique équivalente lors du transfert in vivo de cellules cibles présentant le peptide SSP' Cependant les souris immunisées avec le Melan A LSP présentaient un pourcentage plus élevé de CD8 ayant un Phénotype «centra, memory» (CD44h' CD62L+ and CCR7+ CD62L+) que les souris immunisées avec le SSP, même dix mois après I'immunisation. Par ailleurs, la réponse CD8 au Melan A LSP était strictement dépendante des lymphocytes CD4, contrairement à l'immunisation par le Melan A SSP qui n'était pas affectée. Dans l'ensemble ces résultats suggèrent que la chemokine XCL1 et l'adjuvant QS21 améliorent la réponse CD8 à un long peptide synthétique, favorisant ainsi le développement d'une réponse anti-tumeur mémoire durable. Ces observations pourraient être utiles au développement de nouveau vaccins thérapeutiques contre les tumeurs.

Phospholipid bilayer perturbing-properties underlying lysis induced by pH-sensitive cationic lysine-based surfactants in biomembranes

Many strategies for treating diseases require the delivery of drugs into the cell cytoplasm following internalization within endosomal vesicles. Thus, compounds triggered by low pH to disrupt membranes and release endosomal contents into the cytosol are of particular interest. Here, we report novel cationic lysine-based surfactants (hydrochloride salts of Nε- and Nα-acyl lysine methyl ester) that differ in the position of the positive charge and the length of the alkyl chain. Amino acid-based surfactants could be promising novel biomaterials in drug delivery systems, given their biocompatible properties and low cytotoxic potential. We examined their ability to disrupt the cell membrane in a range of pH values, concentrations and incubation times, using a standard hemolysis assay as a model of endosomal membranes. Furthermore, we addressed the mechanism of surfactant-mediated membrane destabilization, including the effects of each surfactant on erythrocyte morphology as a function of pH. We found that only surfactants with the positive charge on the α-amino group of lysine showed pH-sensitive hemolytic activity and improved kinetics within the endosomal pH range, indicating that the positive charge position is critical for pH-responsive behavior. Moreover, our results showed that an increase in the alkyl chain length from 14 to 16 carbon atoms was associated with a lower ability to disrupt cell membranes. Knowledge on modulating surfactant-lipid bilayer interactions may help us to develop more efficient biocompatible amino acid-based drug delivery devices.

Pom1 gradient buffering through intermolecular auto-phosphorylation.

Concentration gradients provide spatial information for tissue patterning and cell organization, and their robustness under natural fluctuations is an evolutionary advantage. In rod-shaped Schizosaccharomyces pombe cells, the DYRK-family kinase Pom1 gradients control cell division timing and placement. Upon dephosphorylation by a Tea4-phosphatase complex, Pom1 associates with the plasma membrane at cell poles, where it diffuses and detaches upon auto-phosphorylation. Here, we demonstrate that Pom1 auto-phosphorylates intermolecularly, both in vitro and in vivo, which confers robustness to the gradient. Quantitative imaging reveals this robustness through two system's properties: The Pom1 gradient amplitude is inversely correlated with its decay length and is buffered against fluctuations in Tea4 levels. A theoretical model of Pom1 gradient formation through intermolecular auto-phosphorylation predicts both properties qualitatively and quantitatively. This provides a telling example where gradient robustness through super-linear decay, a principle hypothesized a decade ago, is achieved through autocatalysis. Concentration-dependent autocatalysis may be a widely used simple feedback to buffer biological activities.

Anti-MPER antibodies with heterogeneous neutralization capacity are detectable in most untreated HIV-1 infected individuals

Background The MPER region of the HIV-1 envelope glycoprotein gp41 is targeted by broadly neutralizing antibodies. However, the localization of this epitope in a hydrophobic environment seems to hamper the elicitation of these antibodies in HIV infected individuals. We have quantified and characterized anti-MPER antibodies by ELISA and by flow cytometry using a collection of mini gp41-derived proteins expressed on the surface of 293T cells. Longitudinal plasma samples from 35 HIV-1 infected individuals were assayed for MPER recognition and MPER-dependent neutralizing capacity using HIV-2 viruses engrafted with HIV-1 MPER sequences. Results Miniproteins devoid of the cysteine loop of gp41 exposed the MPER on 293T cell membrane. Anti-MPER antibodies were identified in most individuals and were stable when analyzed in longitudinal samples. The magnitude of the responses was strongly correlated with the global response to the HIV-1 envelope glycoprotein, suggesting no specific limitation for anti-MPER antibodies. Peptide mapping showed poor recognition of the C-terminal MPER moiety and a wide presence of antibodies against the 2F5 epitope. However, antibody titers failed to correlate with 2F5-blocking activity and, more importantly, with the specific neutralization of HIV-2 chimeric viruses bearing the HIV-1 MPER sequence; suggesting a strong functional heterogeneity in anti-MPER humoral responses. Conclusions Anti-MPER antibodies can be detected in the vast majority of HIV-1 infected individuals and are generated in the context of the global anti-Env response. However, the neutralizing capacity is heterogeneous suggesting that eliciting neutralizing anti-MPER antibodies by immunization might require refinement of immunogens to skip nonneutralizing responses.

N-acetylcysteine treatment ameliorates the skeletal phenotype of a mouse model of diastrophic dysplasia.

Diastrophic dysplasia (DTD) is a recessive chondrodysplasia caused by mutations in SLC26A2, a cell membrane sulfate-chloride antiporter. Sulfate uptake impairment results in low cytosolic sulfate, leading to cartilage proteoglycan (PG) undersulfation. In this work, we used the dtd mouse model to study the role of N-acetyl-l-cysteine (NAC), a well-known drug with antioxidant properties, as an intracellular sulfate source for macromolecular sulfation. Because of the important pre-natal phase of skeletal development and growth, we administered 30 g/l NAC in the drinking water to pregnant mice to explore a possible transplacental effect on the fetuses. When cartilage PG sulfation was evaluated by high-performance liquid chromatography disaccharide analysis in dtd newborn mice, a marked increase in PG sulfation was observed in newborns from NAC-treated pregnancies when compared with the placebo group. Morphometric studies of the femur, tibia and ilium after skeletal staining with alcian blue and alizarin red indicated a partial rescue of abnormal bone morphology in dtd newborns from treated females, compared with pups from untreated females. The beneficial effect of increased macromolecular sulfation was confirmed by chondrocyte proliferation studies in cryosections of the tibial epiphysis by proliferating cell nuclear antigen immunohistochemistry: the percentage of proliferating cells, significantly reduced in the placebo group, reached normal values in dtd newborns from NAC-treated females. In conclusion, NAC is a useful source of sulfate for macromolecular sulfation in vivo when extracellular sulfate supply is reduced, confirming the potential of therapeutic approaches with thiol compounds to improve skeletal deformity and short stature in human DTD and related disorders.

Structural and functional properties of two human FXYD3 (Mat-8) isoforms.

Six of 7 FXYD proteins have been shown to be tissue-specific modulators of Na,K-ATPase. In this study, we have identified two splice variants of human FXYD3, or Mat-8, in CaCo-2 cells. Short human FXYD3 has 72% sequence identity with mouse FXYD3, whereas long human FXYD3 is identical to short human FXYD3 but has a 26-amino acid insertion after the transmembrane domain. Short and long human FXYD3 RNAs and proteins are differentially expressed during differentiation of CaCo-2 cells. Long human FXYD3 is mainly expressed in nondifferentiated cells and short human FXYD3 in differentiated cells and both FXYD3 variants can be co-immunoprecipitated with a Na,K-ATPase antibody. In contrast to mouse FXYD3, which has two transmembrane domains for lack of cleavage of the signal peptide, human FXYD3 has a cleavable signal peptide and adopts a type I topology. After co-expression in Xenopus oocytes, both human FXYD3 variants associate stably only with Na,K-ATPase isozymes but not with H,K-ATPase or Ca-ATPase. Similar to mouse FXYD3, short human FXYD3 decreases the apparent K(+) and Na(+) affinity of Na,K-ATPase over a large range of membrane potentials. On the other hand, long human FXYD3 decreases the apparent K(+) affinity only at slightly negative and positive membrane potentials and increases the apparent Na(+) affinity of Na,K-ATPase. Finally, both short and long human FXYD3 induce a hyperpolarization activated current, similar to that induced by mouse FXYD3. Thus, we have characterized two human FXYD3 isoforms that are differentially expressed in differentiated and non-differentiated cells and show different functional properties.

Cardiac tissue engineering by electrical stimulation with subcutaneous and cardiac adipose-tissue derived progenitor cells (ATDPCs)

A major challenge of cardiac tissue engineering is directing cells to establish the physiological structure and function of the myocardium being replaced. In native heart, pacing cells generate electrical stimuli that spread throughout the heartcausing cell membrane depolarization and activation of contractile apparatus. We ought to examine whether electricalstimulation of adipose tissue-derived progenitor cells (ATDPCs) exerts phenotypic and genetic changes that enhance theircardiomyogenic potential.

Multiparametric magnetic resonance imaging of the prostate: current concepts

Multiparametric MR (mpMR) imaging is rapidly evolving into the mainstay in prostate cancer (PCa) imaging. Generally, the examination consists of T2-weighted sequences, diffusion-weighted imaging (DWI), dynamic contrast-enhanced (DCE) evaluation, and less often proton MR spectroscopy imaging (MRSI). Those functional techniques are related to biological properties of the tumor, so that DWI correlates to cellularity and Gleason scores, DCE correlates to angiogenesis, and MRSI correlates to cell membrane turnover. The combined use of those techniques enhances the diagnostic confidence and allows for better characterization of PCa. The present article reviews and illustrates the technical aspects and clinical applications of each component of mpMR imaging, in a practical approach from the urological standpoint.

Carcinogenicidade do carbendazim e seus metabólitos

The carcinogenic potential of carbendazim and its metabolites was analyzed using statistical treatment of electronic parameters obtained from DFT/ 6-311++G(d,p) and AM1 calculations. The carcinogen-DNA interaction is described in the framework of the theory of unsynchronized resonance of covalent bond as a process of electron transfer involving the HOMO and LUMO frontier orbitals. Through a Principal Component Analysis (PCA) of the electron affinity, carcinogen-DNA interaction energy, electrostatic attraction and cell membrane permeability (dipole moment m and partition coefficient LogP) evidence was obtained showing carbendazim displays carcinogenic activity. For the metabolites of carbendazim, no evidence was found in the literature of their carcinogenic activities. However, the electronic parameters for these metabolites exhibited similarity to known carcinogens, thereby showing the importance of the results obtained in this study for a policy based on the precautionary principle.

Conformational Regulation of alpha2beta1 Integrin in Ligand Binding

Integrins are heterodimeric cell adhesion receptors involved in cell-cell and cell-extracellular matrix (ECM) interactions. They transmit bidirectional signals across the cell membrane. This results in a wide range of biological events from cell differentiation to apoptosis. alpha2beta1 integrin is an abundant collagen receptor expressed on the surface of several cell types. In addition to ECM ligands, alpha2beta1 integrins are bound by echovirus 1 (EV1) which uses alpha2beta1 as a receptor to initiate its life cycle in the infected cell. The aim of this thesis project was to provide further insight into the mechanisms of alpha2beta1 integrin ligand recognition and receptor activation. Collagen fibrils are the principal tensile elements of the ECM. Yet, the interaction of alpha2beta1 integrin with the fibrillar form of collagen I has received relatively little attention. This research focused on the ability of alpha2beta1 integrin to act as a receptor for type I collagen fibrils. Also the molecular requirements of the EV1 interaction with alpha2beta1 were studied. Conventionally, ligand binding has been suggested to require integrin activation and the binding may further trigger integrin signalling. Another main objective of this study was to elucidate both the inside-out and outside-in signalling mechanisms of alpha2beta1 integrin in adherent cells. The results indicated that alpha2beta1 integrin is the principal integrin-type collagen receptor for type I collagen fibrils, and alpha2beta1 may participate in the regulation of pericellular collagen fibrillogenesis. Furthermore, alpha2beta1 integrin inside-out activation appeared to be synergistically regulated by integrin clustering and conformational activation. The triggering of alpha2beta1 integrin outside-in signalling, however, was shown to require both conformational changes and clustering. In contrast to ECM ligands, EV1 appeared to take advantage of the bent, inactive form of alpha2beta1 integrin in initiating its life cycle in the cell. This research together with other recent studies, has shed light on the molecular mechanisms of integrin activation. It is becoming evident that large ligands are able to bind to the bent form of integrin, which has been previously considered to be physiologically inactive. Consequently, our understanding of the conformational modulation of integrins upon activation is changing.

Expression and distribution of connexin 32 in rat liver with experimentally induced fibrosis

The connexin 32 (Cx32) is a protein that forms the channels that promote the gap junction intercellular communication (GJIC) in the liver, allowing the diffusion of small molecules through cytosol from cell-to-cell. Hepatic fibrosis is characterized by a disruption of normal tissue architeture by cellular lesions, and may alter the GJIC. This work aimed to study the expression and distribution of Cx32 in liver fibrosis induced by the oral administration of dimethylnitrosamine in female Wistar rats. The necropsy of the rats was carried out after five weeks of drug administration. They presented a hepatic fibrosis state. Sections from livers with fibrosis and from control livers were submitted to immunohistochemical, Real Time-PCR and Western-Blot analysis to Cx32. In fibrotic livers the Cxs were diffusely scattered in the cytoplasm, contrasting with the control livers, where the Cx32 formed junction plaques at the cell membrane. Also it was found a decrease in the gene expression of Cx32 without reduction in the protein quantity when compared with controls. These results suggest that there the mechanism of intercellular communication between hepatocytes was reduced by the fibrotic process, which may predispose to the occurrence of a neoplastic process, taken in account that connexins are considered tumor suppressing genes.

Copper accumulation in Xanthomonas campestris pv. vesicatoria

A strain of Xanthomonas campestris pv. vesicatoria showing resistance to 1.2 mM cupric sulfate was analyzed by atomic absorption spectroscopy and ESI (electron spectrophotometry imaging). Accumulation of copper was detected in the periphery of the cell membrane region, suggesting that the mechanism of copper resistance is similar to that previously described for Pseudomonas species. The ESI technique was used to detect copper in the membrane region. Copper-resistance in X. campestris pv. vesicatoria 484 is inducible and occurs by accumulation of the metal and not by efflux mechanism as has been suggested. The growth curve also showed that this system is inducible.

Analysis of viral and cellular parameters which affect the fusion process of influenza viruses

In the present investigation we studied the fusogenic process developed by influenza A, B and C viruses on cell surfaces and different factors associated with virus and cell membrane structures. The biological activity of purified virus strains was evaluated in hemagglutination, sialidase and fusion assays. Hemolysis by influenza A, B and C viruses ranging from 77.4 to 97.2%, from 20.0 to 65.0%, from 0.2 to 93.7% and from 9.0 to 76.1% was observed when human, chicken, rabbit and monkey erythrocytes, respectively, were tested at pH 5.5. At this pH, low hemolysis indexes for influenza A, B and C viruses were observed if horse erythrocytes were used as target cells for the fusion process, which could be explained by an inefficient receptor binding activity of influenza on N-glycolyl sialic acids. Differences in hemagglutinin receptor binding activity due to its specificity to N-acetyl or N-glycolyl cell surface oligosaccharides, density of these cellular receptors and level of negative charges on the cell surface may possibly explain these results, showing influence on the sialidase activity and the fusogenic process. Comparative analysis showed a lack of dependence between the sialidase and fusion activities developed by influenza B viruses. Influenza A viruses at low sialidase titers (<2) also exhibited clearly low hemolysis at pH 5.5 (15.8%), while influenza B viruses with similarly low sialidase titers showed highly variable hemolysis indexes (0.2 to 78.0%). These results support the idea that different virus and cell-associated factors such as those presented above have a significant effect on the multifactorial fusion process

Targeting and translocation of endothelial nitric oxide synthase

This review explores advances in our understanding of the intracellular regulation of the endothelial isoform of nitric oxide synthase (eNOS) in the context of its dynamically regulated subcellular targeting. Nitric oxide (NO) is a labile molecule, and may play important biological roles both within the cell in which it is synthesized and in its interactions with nearby cells and molecules. The localization of eNOS within the cell importantly influences the biological role and chemical fate of the NO produced by the enzyme. eNOS, a Ca2+/calmodulin-dependent enzyme, is subject to a complex pattern of intracellular regulation, including co- and post-translational modifications and interactions with other proteins and ligands. In endothelial cells and cardiac myocytes eNOS is localized in specialized plasmalemmal signal-transducing domains termed caveolae; acylation of the enzyme by the fatty acids myristate and palmitate is required for targeting of the protein to caveolae. Targeting to caveolae facilitates eNOS activation following receptor stimulation. In resting cells, eNOS is tonically inhibited by its interactions with caveolin, the scaffolding protein in caveolae. However, following agonist activation, eNOS dissociates from caveolin, and nearly all the eNOS translocates to structures within the cell cytosol; following more protracted incubations with agonists, most of the cytosolic enzyme subsequently translocates back to the cell membrane. The agonist-induced internalization of eNOS is completely abrogated by chelation of intracellular Ca2+. These rapid receptor-mediated effects are seen not only for "classic" eNOS agonists such as bradykinin, but also for estradiol, indicating a novel non-genomic role for estrogen in eNOS activation. eNOS targeting to the membrane is labile, and is subject to receptor-regulated Ca2+-dependent reversible translocation, providing another point for regulation of NO-dependent signaling in the vascular endothelium.

Abnormal proliferative response of the carotid artery of spontaneously hypertensive rats after angioplasty may be related to the depolarized state of its smooth muscle cells

Hypertension is one of the major precursors of atherosclerotic vascular disease, and vascular smooth muscle abnormal cell replication is a key feature of plaque formation. The present study was conducted to examine the relationship between hypertension and smooth muscle cell proliferation after balloon injury and to correlate neointima formation with resting membrane potential of uninjured smooth muscle cells, since it has been suggested that altered vascular function in hypertension may be related to the resetting of the resting membrane potential in spontaneously hypertensive rats (SHR). Neointima formation was induced by balloon injury to the carotid arteries of SHR and renovascular hypertensive rats (1K-1C), as well as in their normotensive controls, i.e., Wistar Kyoto (WKY) and normal Wistar (NWR) rats. After 14 days the animals were killed and the carotid arteries were submitted to histomorphometric and immunohistochemical analyses. Resting membrane potential measurements showed that uninjured carotid arteries from SHR smooth muscle cells were significantly depolarized (-46.5 ± 1.9 mV) compared to NWR (-69 ± 1.4 mV), NWR 1K-1C (-60.8 ± 1.6 mV), WKY (-67.1 ± 3.2 mV) and WKY 1K-1C (-56.9 ± 1.2 mV). The SHR arteries responded to balloon injury with an enhanced neointima formation (neo/media = 3.97 ± 0.86) when compared to arteries of all the other groups (NWR 0.93 ± 0.65, NWR 1K-1C 1.24 ± 0.45, WKY 1.22 ± 0.32, WKY 1K-1C 1.15 ± 0.74). Our results indicate that the increased fibroproliferative response observed in SHR is not related to the hypertensive state but could be associated with the resetting of the carotid smooth muscle cell resting membrane potential to a more depolarized state.