Novel Titanocene Y derivative with albumin affinity exhibits improved anticancer activity against platinum resistant cells.

The antitumor activity of Ti(IV)-based compounds put them in the spotlight for cancer treatment in the past, but their lack of stability in vivo due to a high rate of hydrolysis has hindered their development as antitumor drugs. As a possible solution for this problem, we have reported a synthesis strategy through which we combined a titanocene fragment, a tridentate ligand, and a long aliphatic chain. This strategy allowed us to generate a titanium compound (Myr-Ti) capable of interacting with albumin, highly stable in water and with cytotoxic activity in tumor cells[1]. Following a similar strategy, now we report the synthesis of a new compound (Myr-TiY) derived from titanocene Y that shows antitumoral activity in a cisplatin resistant model with a 50% inhibitory concentration (IC50) of 41-76 µM. This new compound shows high stability and a strong interaction with human serum albumin. Myr-TiY has a significant antiproliferative and proapoptotic effect on the tested cancer cells and shows potential tumor selectivity when assayed in non-tumor human epithelial cells being more selective (1.3-3.8 times) for tumor cells than cisplatin. These results lead us to think that the described synthesis strategy could be useful to generate compounds for the treatment of both cisplatin-sensitive and cisplatin-resistant cancers.

La presencia de receptores Fc de IgG activadores en macrófagos agrava el desarrollo de aneurisma aórtico abdominal experimental / The presence of activating IgG Fc receptors in macrophages aggravates the development of experimental abdominal aortic aneurysm

Introducción: El aneurisma aórtico abdominal (AAA) es una afección degenerativa y multifactorial caracterizada por una dilatación progresiva de la aorta y activación crónica de inflamación, actividad proteolítica y estrés oxidativo en la pared vascular. La respuesta inmune dependiente de anticuerpos IgG frente a antígenos expuestos en el vaso dañado está implicada en la formación y progresión del AAA, aunque los mecanismos no son del todo conocidos. En este trabajo analizamos la funcionalidad de los receptores Fc de IgG (FcγR), en particular los expresados por el monocito/macrófago, en el desarrollo del AAA experimental. Métodos: En el modelo de AAA inducido por perfusión aórtica de elastasa se examinaron, mediante histología y PCR cuantitativa, las aortas abdominales de ratones de fenotipo salvaje y de ratones deficientes en FcγR, sin y con transferencia adoptiva de macrófagos. In vitro, macrófagos murinos se transfectaron con ARN de interferencia de FcγRIV/CD16.2 o se trataron con un inhibidor de la cinasa Syk antes de la estimulación con inmunocomplejos de IgG. Resultados: La transferencia adoptiva de macrófagos a ratones deficientes en FcγR incrementó su susceptibilidad al desarrollo de AAA. En los ratones que recibieron macrófagos con FcγR funcionales se observó un mayor incremento del diámetro aórtico y del contenido de macrófagos y linfocitos B, así como un aumento en la expresión de la quimiocina CCL2, las citocinas TNF-α e IL-17, la metaloproteinasa MMP2, la enzima prooxidante NADPH oxidasa-2 y las isoformas FcγRIII/CD16 y FcγRIV/CD16.2. In vitro, tanto el silenciamiento génico de FcγRIV/CD16.2 como la inhibición de Syk en macrófagos redujeron la producción de citocinas y anión superóxido inducida por inmunocomplejos...(AU)

Introduction: Abdominal aortic aneurysm (AAA) is a multifactorial, degenerative disease characterized by progressive aortic dilation and chronic activation of inflammation, proteolytic activity, and oxidative stress in the aortic wall. The immune response triggered by antibodies against antigens present in the vascular wall participates in the formation and progression of AAA through mechanisms not completely understood. This work analyses the function of specific IgG receptors (FcγR), especially those expressed by monocytes/macrophages, in the development of experimental AAA. Methods: In the elastase-induced AAA model, the abdominal aortas from wildtype and FcγR deficient mice with/without macrophage adoptive transfer were analysed by histology and quantitative PCR. In vitro, mouse macrophages were transfected with RNA interference of FcγRIV/CD16.2 or treated with Syk kinase inhibitor before stimulation with IgG immune complexes. Results: Macrophage adoptive transfer in FcγR deficient mice increased the susceptibility to AAA development. Mice receiving macrophages with functional FcγR exhibited higher aortic diameter increase, higher content of macrophages and B lymphocytes, and upregulated expression of chemokine CCL2, cytokines (TNF-α and IL-17), metalloproteinase MMP2, prooxidant enzyme NADPH oxidase-2, and the isoforms FcγRIII/CD16 and FcγRIV/CD16.2. In vitro, both FcγRIV/CD16.2 gene silencing and Syk inhibition reduced cytokines and reactive oxygen species production induced by immune complexes in macrophages. Conclusions: Activation of macrophage FcγR contributes to AAA development by inducing mediators of inflammation, proteolysis, and oxidative stress. Modulation of FcγR or effector molecules may represent a potential target for AAA treatment.

The presence of activating IgG Fc receptors in macrophages aggravates the development of experimental abdominal aortic aneurysm. / La presencia de receptores Fc de IgG activadores en macrófagos agrava el desarrollo de aneurisma aórtico abdominal experimental.

INTRODUCTION: Abdominal aortic aneurysm (AAA) is a multifactorial, degenerative disease characterized by progressive aortic dilation and chronic activation of inflammation, proteolytic activity, and oxidative stress in the aortic wall. The immune response triggered by antibodies against antigens present in the vascular wall participates in the formation and progression of AAA through mechanisms not completely understood. This work analyses the function of specific IgG receptors (FcγR), especially those expressed by monocytes/macrophages, in the development of experimental AAA. METHODS: In the elastase-induced AAA model, the abdominal aortas from wildtype and FcγR deficient mice with/without macrophage adoptive transfer were analysed by histology and quantitative PCR. In vitro, mouse macrophages were transfected with RNA interference of FcγRIV/CD16.2 or treated with Syk kinase inhibitor before stimulation with IgG immune complexes. RESULTS: Macrophage adoptive transfer in FcγR deficient mice increased the susceptibility to AAA development. Mice receiving macrophages with functional FcγR exhibited higher aortic diameter increase, higher content of macrophages and B lymphocytes, and upregulated expression of chemokine CCL2, cytokines (TNF-α and IL-17), metalloproteinase MMP2, prooxidant enzyme NADPH oxidase-2, and the isoforms FcγRIII/CD16 and FcγRIV/CD16.2. In vitro, both FcγRIV/CD16.2 gene silencing and Syk inhibition reduced cytokines and reactive oxygen species production induced by immune complexes in macrophages. CONCLUSIONS: Activation of macrophage FcγR contributes to AAA development by inducing mediators of inflammation, proteolysis, and oxidative stress. Modulation of FcγR or effector molecules may represent a potential target for AAA treatment.

Fcγ receptor activation mediates vascular inflammation and abdominal aortic aneurysm development.

BACKGROUND: Abdominal aortic aneurysm (AAA), a degenerative vascular pathology characterized by permanent dilation of the aorta, is considered a chronic inflammatory disease involving innate/adaptive immunity. However, the functional role of antibody-dependent immune response against antigens present in the damaged vessel remains unresolved. We hypothesized that engagement of immunoglobulin G (IgG) Fc receptors (FcγR) by immune complexes (IC) in the aortic wall contributes to AAA development. We therefore evaluated FcγR expression in AAA lesions and analysed whether inhibition of FcγR signaling molecules (γ-chain and Syk kinase) influences AAA formation in mice. METHODS: FcγR gene/protein expression was assessed in human and mouse AAA tissues. Experimental AAA was induced by aortic elastase perfusion in wild-type (WT) mice and γ-chain knockout (γKO) mice (devoid of activating FcγR) in combination with macrophage adoptive transfer or Syk inhibitor treatment. To verify the mechanisms of FcγR in vitro, vascular smooth muscle cells (VSMC) and macrophages were stimulated with IgG IC. RESULTS: FcγR overexpression was detected in adventitia and media layers of human and mouse AAA. Elastase-perfused γKO mice exhibited a decrease in AAA incidence, aortic dilation, elastin degradation, and VSMC loss. This was associated with (1) reduced infiltrating leukocytes and immune deposits in AAA lesions, (2) inflammatory genes and metalloproteinases downregulation, (3) redox balance restoration, and (4) converse phenotype of anti-inflammatory macrophage M2 and contractile VSMC. Adoptive transfer of FcγR-expressing macrophages aggravated aneurysm in γKO mice. In vitro, FcγR deficiency attenuated inflammatory gene expression, oxidative stress, and phenotypic switch triggered by IC. Additionally, Syk inhibition prevented IC-mediated cell responses, reduced inflammation, and mitigated AAA formation. CONCLUSION: Our findings provide insight into the role and mechanisms mediating IgG-FcγR-associated inflammation and aortic wall injury in AAA, which might represent therapeutic targets against AAA disease.

Cyclic mimetics of kinase-inhibitory region of Suppressors of Cytokine Signaling 1: Progress toward novel anti-inflammatory therapeutics.

Herein we investigated the structural and cellular effects ensuing from the cyclization of a potent inhibitor of JAK2 as mimetic of SOCS1 protein, named PS5. The introduction of un-natural residues and a lactam internal bridge, within SOCS1-KIR motif, produced candidates that showed high affinity toward JAK2 catalytic domain. By combining CD, NMR and computational studies, we obtained valuable models of the interactions of two peptidomimetics of SOCS1 to deepen their functional behaviors. Notably, when assayed for their biological cell responses mimicking SOCS1 activity, the internal cyclic PS5 analogues demonstrated able to inhibit JAK-mediated tyrosine phosphorylation of STAT1 and to reduce cytokine-induced proinflammatory gene expression, oxidative stress generation and cell migration. The present study well inserts in the field of low-molecular-weight proteomimetics with improved longtime cellular effects and adds a new piece to the puzzled way for the conversion of bioactive peptides into drugs.

Protective effect of suppressor of cytokine signalling 1-based therapy in experimental abdominal aortic aneurysm.

BACKGROUND AND PURPOSE: Abdominal aortic aneurysm (AAA) is a multifactorial disease characterized by chronic inflammation, oxidative stress and proteolytic activity in the aortic wall. Targeting JAK/signal transducer and activator of transcription (JAK/STAT) pathway is a promising strategy for chronic inflammatory diseases. We investigated the vasculo-protective role of suppressor of cytokine signalling-1 (SOCS1), the negative JAK/STAT regulator, in experimental AAA. EXPERIMENTAL APPROACH: A synthetic, cell permeable peptide (S1) mimic of SOCS1 kinase inhibitory domain to suppress STAT activation was evaluated in the well-established mouse model of elastase-induced AAA by monitoring changes in aortic diameter, cellular composition and gene expression in abdominal aorta. S1 function was further evaluated in cultured vascular smooth muscle cells (VSMC) and macrophages exposed to elastase or elastin-derived peptides. KEY RESULTS: S1 peptide prevented AAA development, evidenced by reduced incidence of AAA, aortic dilation and elastin degradation, partial restoration of medial VSMC and decreased inflammatory cells and oxidative stress in AAA tissue. Mechanistically, S1 suppressed STAT1/3 activation in aorta, down-regulated cytokines, metalloproteinases and altered the expression of cell differentiation markers by favouring anti-inflammatory M2 macrophage and contractile VSMC phenotypes. In vitro, S1 suppressed the expression of inflammatory and oxidative genes, reduced cell migration and reversed the phenotypic switch of macrophages and VSMC. By contrast, SOCS1 silencing promoted inflammatory response. CONCLUSION AND IMPLICATIONS: This preclinical study demonstrates the therapeutic potential of SOCS1-derived peptide to halt AAA progression by suppressing JAK/STAT-mediated inflammation and aortic dilation. S1 peptide may therefore be a valuable option for the treatment of AAA.

Antioxidant Effects of PS5, a Peptidomimetic of Suppressor of Cytokine Signaling 1, in Experimental Atherosclerosis.

The chronic activation of the Janus kinase/signal transducer and activator of the transcription (JAK/STAT) pathway is linked to oxidative stress, inflammation and cell proliferation. Suppressors of cytokine signaling (SOCS) proteins negatively regulate the JAK/STAT, and SOCS1 possesses a small kinase inhibitory region (KIR) involved in the inhibition of JAK kinases. Several studies showed that KIR-SOCS1 mimetics can be considered valuable therapeutics in several disorders (e.g., diabetes, neurological disorders and atherosclerosis). Herein, we investigated the antioxidant and atheroprotective effects of PS5, a peptidomimetic of KIR-SOCS1, both in vitro (vascular smooth muscle cells and macrophages) and in vivo (atherosclerosis mouse model) by analyzing gene expression, intracellular O2•- production and atheroma plaque progression and composition. PS5 was revealed to be able to attenuate NADPH oxidase (NOX1 and NOX4) and pro-inflammatory gene expression, to upregulate antioxidant genes and to reduce atheroma plaque size, lipid content and monocyte/macrophage accumulation. These findings confirm that KIR-SOCS1-based drugs could be excellent antioxidant agents to contrast atherosclerosis.

Targeting NF-κB by the Cell-Permeable NEMO-Binding Domain Peptide Improves Albuminuria and Renal Lesions in an Experimental Model of Type 2 Diabetic Nephropathy.

Diabetic nephropathy (DN) is a multifactorial disease characterized by hyperglycemia and close interaction of hemodynamic, metabolic and inflammatory factors. Nuclear factor-κB (NF-κB) is a principal matchmaker linking hyperglycemia and inflammation. The present work investigates the cell-permeable peptide containing the inhibitor of kappa B kinase γ (IKKγ)/NF-κB essential modulator (NEMO)-binding domain (NBD) as therapeutic option to modulate inflammation in a preclinical model of type 2 diabetes (T2D) with DN. Black and tan, brachyuric obese/obese mice were randomized into 4 interventions groups: Active NBD peptide (10 and 6 µg/g body weight); Inactive mutant peptide (10 µg/g); and vehicle control. In vivo/ex vivo fluorescence imaging revealed efficient delivery of NBD peptide, systemic biodistribution and selective renal metabolization. In vivo administration of active NBD peptide improved albuminuria (>40% reduction on average) and kidney damage, decreased podocyte loss and basement membrane thickness, and modulated the expression of proinflammatory and oxidative stress markers. In vitro, NBD blocked IKK-mediated NF-κB induction and target gene expression in mesangial cells exposed to diabetic-like milieu. These results constitute the first nephroprotective effect of NBD peptide in a T2D mouse model that recapitulates the kidney lesions observed in DN patients. Targeting IKK-dependent NF-κB activation could be a therapeutic strategy to combat kidney inflammation in DN.

Chimeric Peptidomimetics of SOCS 3 Able to Interact with JAK2 as Anti-inflammatory Compounds.

The immunomodulatory effects of Suppressor of Cytokine Signaling (SOCS) proteins, that control the JAK/STAT pathway, indicate them as attractive candidates for immunotherapies. Recombinant SOCS3 protein suppresses the effects of inflammation, and its deletion in neurons or in immune cells increases pathological blood vessels growth. Recently, on the basis of the structure of the ternary complex among SOCS3, JAK2, and gp130, we focused on SOCS3 interfacing regions and designed several interfering peptides (IPs) that were able to mimic SOCS3 biological role in triple negative breast cancer (TNBC) models. Herein, to explore other protein regions involved in JAK2 recognition, several new chimeric peptides connecting noncontiguous SOCS3 regions and including a strongly aromatic fragment were investigated. Their ability to recognize the catalytic domain of JAK2 was evaluated through MST (microscale thermophoresis), and the most promising compound, named KIRCONG chim, exhibited a low micromolar value for dissociation constant. The conformational features of chimeric peptides were analyzed through circular dichroism and NMR spectroscopies, and their anti-inflammatory effects were assessed in cell cultures. Overall data suggest the importance of aromatic contribution in the recognition of JAK2 and that SOCS3 peptidomimetics could be endowed with a therapeutic potential in diseases with activated inflammatory cytokines.

Nrf2 Activation Provides Atheroprotection in Diabetic Mice Through Concerted Upregulation of Antioxidant, Anti-inflammatory, and Autophagy Mechanisms.

Interactive relationships between metabolism, inflammation, oxidative stress, and autophagy in the vascular system play a key role in the pathogenesis of diabetic cardiovascular disease. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a stress-sensitive guarantor of cellular homeostasis, which cytoprotective contributions extend beyond the antioxidant defense. We investigated the beneficial effects and underlying mechanisms of the Nrf2 inducer tert-butyl hydroquinone (tBHQ) on diabetes-driven atherosclerosis. In the experimental model of streptozotocin-induced diabetes in apolipoprotein E-deficient mice, treatment with tBHQ increased Nrf2 activity in macrophages and vascular smooth muscle cells within atherosclerotic lesions. Moreover, tBHQ significantly decreased the size, extension and lipid content of atheroma plaques, and attenuated inflammation by reducing lesional macrophages (total number and M1/M2 phenotype balance), foam cell size and chemokine expression. Atheroprotection was accompanied by both systemic and local antioxidant effects, characterized by lower levels of superoxide anion and oxidative DNA marker 8-hydroxy-2'-deoxyguanosine, reduced expression of NADPH oxidase subunits, and increased antioxidant capacity. Interestingly, tBHQ treatment upregulated the gene and protein expression of autophagy-related molecules and also enhanced autophagic flux in diabetic mouse aorta. In vitro, Nrf2 activation by tBHQ suppressed cytokine-induced expression of pro-inflammatory and oxidative stress genes, altered macrophage phenotypes, and promoted autophagic activity. Our results reinforce pharmacological Nrf2 activation as a promising atheroprotective approach in diabetes, according to the plethora of cytoprotective mechanisms involved in the resolution of inflammation and oxidative stress, and restoring autophagy.

SOCS1-targeted therapy ameliorates renal and vascular oxidative stress in diabetes via STAT1 and PI3K inhibition.

Oxidative stress resulting from excessive production of reactive oxygen species (ROS) or impaired antioxidant defenses is closely related to the development of diabetic vascular complications, including nephropathy and atherosclerosis. Chronic activation of Janus kinase/Signal transducer and activator of transcription (JAK/STAT) signaling pathway contributes to diabetic complications by inducing expression of genes involved in cell proliferation, fibrosis, inflammation, and oxidative stress. Suppressors of cytokine signaling (SOCS) family of endogenous JAK/STAT regulators is an attractive target for therapeutic intervention. We investigated the beneficial effect of two different SOCS1-targeted therapies (adenovirus-mediated gene transfer and kinase-inhibitory region peptidomimetic) to combat oxidative stress injury in an experimental diabetes model of concomitant renal and macrovascular disease (streptozotocin-induced diabetic apolipoprotein E-deficient mouse). Diabetes resulted in progressive alteration of redox balance in mice, as demonstrated by increased ROS levels and decreased antioxidant activity, which ultimately led to renal dysfunction and vascular injury. The molecular and pathological alterations in early diabetes were partially reversed by preventive intervention with SOCS1-targeted therapies. Importantly, SOCS1 peptidomimetic provided reno- and atheroprotection in diabetic mice even in a setting of established disease. Compared with untreated controls, kidney and aorta from SOCS1-treated mice exhibited significantly lower levels of superoxide anion, DNA oxidation marker and NADPH oxidase (Nox) subunits, along with higher expression of antioxidant enzymes. These trends correlated with a reduction in parameters of renal damage (albuminuria, creatinine and tubular injury), atherosclerosis (lesion size) and inflammation (leukocytes and chemokines). Mechanistic studies in renal, vascular and phagocytic cells exposed to cytokines and high-glucose showed that SOCS1 blocked ROS generation by inhibiting both Nox complex assembly and Nox subunit expression, an effect mediated by inactivation of JAK2, STAT1, and PI3K signaling pathways. This study provides evidence for SOCS1-targeted therapies, especially SOCS1 peptidomimetic, as an alternative antioxidant strategy to limit the progression of diabetic micro- and macrovascular complications.

Structure-activity studies of peptidomimetics based on kinase-inhibitory region of suppressors of cytokine signaling 1.

Suppressors of Cytokine Signaling (SOCS) proteins are negative regulators of JAK proteins that are receptor-associated tyrosine kinases, which play key roles in the phosphorylation and subsequent activation of several transcription factors named STATs. Unlike the other SOCS proteins, SOCS1 and 3 show, in the N-terminal portion, a small kinase inhibitory region (KIR) involved in the inhibition of JAK kinases. Drug discovery processes of compounds based on KIR sequence demonstrated promising in functional in vitro and in inflammatory animal models and we recently developed a peptidomimetic called PS5, as lead compound. Here, we investigated the cellular ability of PS5 to mimic SOCS1 biological functions in vascular smooth muscle cells and simultaneously we set up a new binding assay for the screening and identification of JAK2 binders based on a SPR experiment that revealed more robust with respect to previous ELISAs. On this basis, we designed several peptidomimetics bearing new structural constraints that were analyzed in both affinities toward JAK2 and conformational features through Circular Dichroism and NMR spectroscopies. Introduced chemical modifications provided an enhancement of serum stabilities of new sequences that could aid the design of future mimetic molecules of SOCS1 as novel anti-inflammatory compounds.

Interplay between HSP90 and Nrf2 pathways in diabetes-associated atherosclerosis / Interacción entre las vías HSP90 y Nrf2 en la aterosclerosis asociada a diabetes

Introduction: Oxidative stress and inflammation are determinant processes in the development of diabetic vascular complications. Heat shock protein 90 (HSP90) overexpression in atherosclerotic plaques plays a role in sustaining inflammatory mechanisms, and its specific inhibition prevents atherosclerosis. The present work investigates, in a mouse model of diabetes-driven atherosclerosis, whether atheroprotection by pharmacological HSP90 inhibition is accomplished by bolstering antioxidant defense mechanisms headed by nuclear factor erythroid-derived 2-like 2 (Nrf2). Methods: Streptozotocin-induced diabetic apolipoprotein E-deficient mice were randomized to receive vehicle or HSP90 inhibitor (17-dimethylaminoethylamino-17-demethoxygeldanamycin, 4mg/kg) for 10 weeks. Aortic root sections were analyzed for plaque size and composition, transcription factor activity, and expression of inflammatory and antioxidant markers. In vitro studies were performed in murine macrophages cultured under hyperglycemic conditions. Results: Treatment with HSP90 inhibitor promoted the activation of Nrf2 in the aortic tissue of diabetic mice (predominantly localized in macrophages and smooth muscle cells) and also in cultured cells. Nrf2 induction was associated with a concomitant inhibition of nuclear factor-κB (NF-κB) in atherosclerotic plaques, thus resulting in a significant reduction in lesion size and inflammatory component (leukocytes and cytokines). Furthermore, atheroprotection by HSP90 inhibition was linked to the induction of cytoprotective HSP70, antioxidant enzymes (heme oxygenase-1, superoxide dismutase and catalase) and autophagy machinery (LC3 and p62/SQSTM1) in aortic tissue. Conclusion: HSP90 inhibition protects from atherosclerosis in experimental diabetes through the induction of Nrf2-dependent cytoprotective mechanisms, reinforcing its therapeutic potential

Introducción: Estrés oxidativo e inflamación son procesos clave en las complicaciones vasculares de la diabetes. La expresión de heat shock protein 90 (HSP90) en placas ateroscleróticas prolonga la inflamación, y su inhibición previene la aterosclerosis. Este trabajo investiga, en un modelo de aterosclerosis asociada a diabetes, si el efecto ateroprotector de la inhibición farmacológica de HSP90 está mediado por la inducción de mecanismos de defensa antioxidante dependientes del factor de transcripción nuclear factor erythroid-derived 2-like 2 (Nrf2). Métodos: Ratones deficientes en apolipoproteína E con diabetes por estreptozotocina se trataron durante 10 semanas con el inhibidor de HSP90 (17-dimetilaminoetilamino-demetoxigeldanamicina, 4mg/kg). Analizamos tamaño/composición de las placas, factores de transcripción y marcadores inflamatorios y antioxidantes. In vitro estudiamos macrófagos murinos bajo condiciones de hiperglucemia. Resultados: El inhibidor de HSP90 indujo activación de Nrf2 en aorta de ratones diabéticos (localizado en macrófagos y células de músculo liso) y en células en cultivo. La activación de Nrf2 junto con la inhibición de nuclear factor-κB (NF-κB) en las placas ateroscleróticas resultó en una reducción significativa del tamaño de la lesión y del componente inflamatorio (leucocitos y citocinas). Este efecto ateroprotector se vinculó con un aumento de la proteína citoprotectora HSP70, enzimas antioxidantes (hemoxigenasa-1, superóxido dismutasa y catalasa) y genes de autofagia (LC3 y p62/SQSTM1) en aorta de ratones tratados con el inhibidor de HSP90. Conclusión: La inhibición de HSP90 protege del desarrollo de aterosclerosis en la diabetes experimental a través de la inducción de mecanismos citoprotectores dependientes de Nrf2, lo que refuerza su potencial terapéutico

Interplay between HSP90 and Nrf2 pathways in diabetes-associated atherosclerosis.

INTRODUCTION: Oxidative stress and inflammation are determinant processes in the development of diabetic vascular complications. Heat shock protein 90 (HSP90) overexpression in atherosclerotic plaques plays a role in sustaining inflammatory mechanisms, and its specific inhibition prevents atherosclerosis. The present work investigates, in a mouse model of diabetes-driven atherosclerosis, whether atheroprotection by pharmacological HSP90 inhibition is accomplished by bolstering antioxidant defense mechanisms headed by nuclear factor erythroid-derived 2-like 2 (Nrf2). METHODS: Streptozotocin-induced diabetic apolipoprotein E-deficient mice were randomized to receive vehicle or HSP90 inhibitor (17-dimethylaminoethylamino-17-demethoxygeldanamycin, 4mg/kg) for 10 weeks. Aortic root sections were analyzed for plaque size and composition, transcription factor activity, and expression of inflammatory and antioxidant markers. In vitro studies were performed in murine macrophages cultured under hyperglycemic conditions. RESULTS: Treatment with HSP90 inhibitor promoted the activation of Nrf2 in the aortic tissue of diabetic mice (predominantly localized in macrophages and smooth muscle cells) and also in cultured cells. Nrf2 induction was associated with a concomitant inhibition of nuclear factor-κB (NF-κB) in atherosclerotic plaques, thus resulting in a significant reduction in lesion size and inflammatory component (leukocytes and cytokines). Furthermore, atheroprotection by HSP90 inhibition was linked to the induction of cytoprotective HSP70, antioxidant enzymes (heme oxygenase-1, superoxide dismutase and catalase) and autophagy machinery (LC3 and p62/SQSTM1) in aortic tissue. CONCLUSION: HSP90 inhibition protects from atherosclerosis in experimental diabetes through the induction of Nrf2-dependent cytoprotective mechanisms, reinforcing its therapeutic potential.

Suppressor of Cytokine Signaling-1 Peptidomimetic Limits Progression of Diabetic Nephropathy.

Diabetes is the main cause of CKD and ESRD worldwide. Chronic activation of Janus kinase and signal transducer and activator of transcription (STAT) signaling contributes to diabetic nephropathy by inducing genes involved in leukocyte infiltration, cell proliferation, and extracellular matrix accumulation. This study examined whether a cell-permeable peptide mimicking the kinase-inhibitory region of suppressor of cytokine signaling-1 (SOCS1) regulatory protein protects against nephropathy by suppressing STAT-mediated cell responses to diabetic conditions. In a mouse model combining hyperglycemia and hypercholesterolemia (streptozotocin diabetic, apoE-deficient mice), renal STAT activation status correlated with the severity of nephropathy. Notably, compared with administration of vehicle or mutant inactive peptide, administration of the SOCS1 peptidomimetic at either early or advanced stages of diabetes ameliorated STAT activity and resulted in reduced serum creatinine level, albuminuria, and renal histologic changes (mesangial expansion, tubular injury, and fibrosis) over time. Mice treated with the SOCS1 peptidomimetic also exhibited reduced kidney leukocyte recruitment (T lymphocytes and classic M1 proinflammatory macrophages) and decreased expression levels of proinflammatory and profibrotic markers that were independent of glycemic and lipid changes. In vitro, internalized peptide suppressed STAT activation and target gene expression induced by inflammatory and hyperglycemic conditions, reduced migration and proliferation in mesangial and tubuloepithelial cells, and altered the expression of cytokine-induced macrophage polarization markers. In conclusion, our study identifies SOCS1 mimicking as a feasible therapeutic strategy to halt the onset and progression of renal inflammation and fibrosis in diabetic kidney disease.

The Tetraspanin TSPAN33 Controls TLR-Triggered Macrophage Activation through Modulation of NOTCH Signaling.

The involvement of NOTCH signaling in macrophage activation by Toll receptors has been clearly established, but the factors and pathways controlling NOTCH signaling during this process have not been completely delineated yet. We have characterized the role of TSPAN33, a tetraspanin implicated in a disintegrin and metalloproteinase (ADAM) 10 maturation, during macrophage proinflammatory activation. Tspan33 expression increases in response to TLR signaling, including responses triggered by TLR4, TLR3, and TLR2 activation, and it is enhanced by IFN-γ. In this study, we report that induction of Tspan33 expression by TLR and IFN-γ is largely dependent on NOTCH signaling, as its expression is clearly diminished in macrophages lacking Notch1 and Notch2 expression, but it is enhanced after overexpression of a constitutively active intracellular domain of NOTCH1. TSPAN33 is the member of the TspanC8 tetraspanin subgroup more intensely induced during macrophage activation, and its overexpression increases ADAM10, but not ADAM17, maturation. TSPAN33 favors NOTCH processing at the membrane by modulating ADAM10 and/or Presenilin1 activity, thus increasing NOTCH signaling in activated macrophages. Moreover, TSPAN33 modulates TLR-induced proinflammatory gene expression, at least in part, by increasing NF-κB-dependent transcriptional activity. Our results suggest that TSPAN33 represents a new control element in the development of inflammation by macrophages that could constitute a potential therapeutic target.