Carla Iacobini

Accelerated diabetic glomerulopathy in galectin-3/AGE receptor 3 knockout mice

Several molecules were shown to bind advanced glycation end products (AGEs) in vitro, but it is not known whether they all serve as AGE receptors and which functional role they play in vivo. We investigated the role of galectin‐3, a multifunctional lectin with (anti)adhesive and growth‐regulating properties, as an AGE receptor and its contribution to the development of diabetic glomerular disease, using a knockout mouse model. Galectin‐3 knockout mice obtained by gene ablation and the corresponding wild‐type mice were rendered diabetic with streptozotocin and killed 4 months later, together with age‐matched nondiabetic controls. Despite a comparable degree of metabolic derangement, galectin‐3‐deficient mice developed ac‐celerated glomerulopathy vs. the wild‐type animals, as evidenced by the more pronounced increase in protein‐uria, extracellular matrix gene expression, and mesan‐gial expansion. This was associated with a more marked renal/glomerular AGE accumulation, indicating it was attributable to the lack of galectin‐3 AGE receptor function. The galectin‐3‐deficient genotype was associated with reduced expression of receptors implicated in AGE removal (macrophage scavenger receptor A and AGE‐R1) and increased expression of those mediating cell activation (RAGE and AGE‐R2). These results show that the galectin‐3‐regulated AGE receptor pathway is operating in vivo and protects toward AGE‐induced tissue injury in contrast to that through RAGE.—Pugliese, G., Pricci, F., Iacobini, C., Leto, G., Amadio, L., Barsotti, P., Frigeri L., Hsu, D. K., Vlassara, H., Liu, F.‐T., Di Mario, U. Accelerated diabetic glomerulopathy in galectin‐3/AGE receptor 3 knockout mice. FASEB J. 15, 2471–2479 (2001)

Deletion of p66Shc Longevity Gene Protects Against Experimental Diabetic Glomerulopathy by Preventing Diabetes-Induced Oxidative Stress

p66Shc regulates both steady-state and environmental stress-dependent reactive oxygen species (ROS) generation. Its deletion was shown to confer resistance to oxidative stress and protect mice from aging-associated vascular disease. This study was aimed at verifying the hypothesis that p66Shc deletion also protects from diabetic glomerulopathy by reducing oxidative stress. Streptozotocin-induced diabetic p66Shc knockout (KO) mice showed less marked changes in renal function and structure, as indicated by the significantly lower levels of proteinuria, albuminuria, glomerular sclerosis index, and glomerular and mesangial areas. Glomerular content of fibronectin and collagen IV was also lower in diabetic KO versus wild-type mice, whereas apoptosis was detected only in diabetic wild-type mice. Serum and renal tissue advanced glycation end products and plasma isoprostane 8-epi-prostaglandin F2α levels and activation of nuclear factor κB (NF-κB) were also lower in diabetic KO than in wild-type mice. Mesangial cells from KO mice grown under high-glucose conditions showed lower cell death rate, matrix production, ROS levels, and activation of NF-κB than those from wild-type mice. These data support a role for oxidative stress in the pathogenesis of diabetic glomerulopathy and indicate that p66Shc is involved in the molecular mechanism(s) underlying diabetes-induced oxidative stress and oxidant-dependent renal injury.

Tissue Inhibitor of Metalloproteinase 3 Deficiency Causes Hepatic Steatosis and Adipose Tissue Inflammation in Mice

BACKGROUND & AIMS Obesity-driven, low-grade inflammation affects systemic metabolic function and can lead to insulin resistance, hepatic steatosis, and atherosclerosis. Decreased expression of tissue inhibitor of metalloproteinase 3 (Timp3) is a catalyst for insulin resistance and inflammation. Timp3 is a natural inhibitor of matrix metalloproteinases, tumor necrosis factor-alpha-converting enzyme (TACE), and vascular endothelial growth factor receptor 2, and therefore could affect signaling processes involved in inflammation and angiogenesis. METHODS We assessed the effects of Timp3 on inflammation, tissue remodeling, and intermediary metabolism in mice, under conditions of environmental stress (high-fat diet), genetic predisposition to insulin resistance (insulin receptor [Insr] haploinsufficiency), and varying levels of inflammation (Timp3 or Tace deficiencies). Metabolic tests, immunohistochemistry, real-time polymerase chain reaction, and immunoblotting were used to compare data from wild-type, Insr(+/-), Timp3(-/-), Insr(+/-)Timp3(-/-), and Insr(+/-)Tace(+/-) mice placed on high-fat diets for 10 weeks. RESULTS Insr(+/-)Timp3(-/-) mice showed a higher degree of adipose and hepatic inflammation compared with wild-type, Insr(+/-), Timp3(-/-), and Insr(+/-)Tace(+/-) mice. In particular, the Insr(+/-)Timp3(-/-) mice developed macrovesicular steatosis and features of severe nonalcoholic fatty liver disease, including lobular and periportal inflammation, hepatocellular ballooning, and perisinusoidal fibrosis. These were associated with increased expression of inflammatory and steatosis markers, including suppressor of cytokine signaling 3 and stearoyl CoA desaturase 1, in both liver and adipose tissue. Interestingly, Insr(+/-)Tace(+/-) mice had a nearly opposite phenotype. CONCLUSIONS Timp3, possibly through its regulation of TACE, appears to have a role in the pathogenesis of fatty liver disease associated with obesity.

Galectin-3/AGE-receptor 3 knockout mice show accelerated AGE-induced glomerular injury: evidence for a protective role of galectin-3 as an AGE receptor

We previously showed that mice lacking galectin‐3/AGE‐receptor 3 develop accelerated diabetic glomerulopathy. To further investigate the role of galectin‐3/AGE‐receptor function in the pathogenesis of diabetic renal disease, galectin‐3 knockout (KO) and coeval wild‐type (WT) mice were injected for 3 months with 30 μg/day of Nε‐carboxymethyllysine (CML)‐modified or unmodified mouse serum albumin (MSA). Despite receiving equal doses of CML, KO had higher circulating and renal AGE levels and showed more marked renal functional and structural changes than WT mice, with significantly higher proteinuria, albuminuria, glomerular, and mesangial area and glomerular sclerosis index. Renal 4‐hydroxy‐2‐nonenal content and NFκB activation were also more pronounced in KO‐CML vs. WT‐CML. Kidney mRNA levels of fibronectin, laminin, collagen IV, and TGF‐β were up‐regulated, whereas those of matrix metalloproteinase‐2 and ‐14 were down‐regulated, again more markedly in KO‐CML than WT‐CML mice. Basal and CML‐induced RAGE and 80K‐H mRNA levels were higher in KO vs. WT mice. MSA injection did not produce any significant effect in both genotypes. The association of galectin‐3 ablation with enhanced susceptibility to AGE‐induced renal disease, increased AGE levels and signaling, and altered AGE‐receptor pattern indicates that galectin‐3 is operating in vivo as an AGE receptor to afford protection toward AGE‐dependent tissue injury.

Role of Galectin-3 in Diabetic Nephropathy

The advanced glycosylation end products (AGE) participate in the pathogenesis of nephropathy and other diabetic complications through several mechanisms, including their binding to cell surface receptors. The AGE receptors include RAGE, the macrophage scavenger receptors, OST-48 (AGE-R1), 80K-H (AGE-R2), and galectin-3 (AGE-R3). Galectin-3 interacts with the beta-galactoside residues of cell surface and matrix glycoproteins via the carbohydrate recognition domain and with intracellular proteins via peptide-peptide associations mediated by its N-terminus domain. These structural properties enable galectin-3 to exert multiple functions, including the mRNA splicing activity, the control of cell cycle, the regulation of cell adhesion, the modulation of allergic reactions, and the binding of AGE. The lack of transmembrane anchor sequence or signal peptide suggests that it is associated with other AGE receptors, possibly AGE-R1 and AGE-R2, to form an AGE-receptor complex, rather than playing an independent role. In target tissues of diabetic vascular complications, such as the endothelium and mesangium, galectin-3 is weakly expressed under basal conditions and is markedly upregulated by the diabetic milieu (and to a lesser extent by aging). Galectin-3-deficient mice were found to develop accelerated diabetic glomerulopathy versus the wild-type animals, as evidenced by the more pronounced increase in proteinuria, mesangial expansion, and matrix gene expression. This was associated with a more marked renal/glomerular AGE accumulation, suggesting that it was attributable to the lack of galectin-3 AGE-receptor function. These data indicate that galectin-3 is upregulated under diabetic conditions and is operating in vivo to provide protection toward AGE-induced tissue injury, as opposed to RAGE.

Identification of a novel partner of RNA polymerase II subunit 11, Che-1, which interacts with and affects the growth suppression function of Rb

hRPB11 is a core subunit of RNA polymerase II (pol II) specifically down‐regulated on doxorubicin (dox) treatment. Levels of this protein profoundly affect cell differentiation, cell proliferation, and tumorigenicity in vivo. Here we describe Che‐1, a novel human protein that interacts with hRPB11. Che‐1 possesses a domain of high homol‐ogy with Escherichia coli RNA polymerase σ‐factor 70 and SV40 large T antigen. In addition, we report that Che‐1 interacts with the retinoblastoma susceptibility gene (Rb) by two distinct domains. Functionally, we demonstrate that Che‐1 represses the growth suppression function of Rb, counteracting the inhibitory action of Rb on the irans‐activation function of E2F1. These results identify a novel protein that binds Rb and the core of pol II, and suggest that Che‐1 may be part of transcription regulatory complex.—Fanciulli, M., Bruno, T, Di Padova, M., De Angelis, R., Iezzi, S., Iacobini, C, Floridi, A., Passananti, C. Identification of a novel partner of RNA polymerase II subunit 11, Che‐1, which interacts with and affects the growth suppression function of Rb. FASEB J. 14, 904–912 (2000)

Oxidative stress in diabetes-induced endothelial dysfunction involvement of nitric oxide and protein kinase C.

Reactive oxygen species (ROS) formation plays a major role in diabetes-induced endothelial dysfunction, though the molecular mechanism(s) involved and the contribution of nitric oxide (NO) are still unclear. This study using bovine retinal endothelial cells was aimed at assessing (i) the role of oxygen-dependent vs. NO-dependent oxidative stress in the endothelial cell permeability alterations induced by the diabetic milieu and (ii) whether protein kinase C (PKC) activation ultimately mediates these changes. Superoxide, lipid peroxide, and PKC activity were higher under high glucose (HG) vs. normal glucose throughout the 30 d period. Nitrite/nitrate and endothelial NO synthase levels increased at 1 d and decreased thereafter. Changes in monolayer permeability to 125I-BSA induced by 1 or 30 d incubation in HG or exposure to advanced glycosylation endproduct were reduced by treatment with antioxidants or PKC inhibitors, whereas NO blockade prevented only the effect of 1 d HG. HG-induced changes were mimicked by a PKC activator, a superoxide generating system, an NO and superoxide donor, or peroxynitrite (attenuated by PKC inhibition), but not a NO donor. The short-term effect of HG depends on a combined oxidative and nitrosative stress with peroxynitrite formation, whereas the long-term effect is related to ROS generation; in both cases, PKC ultimately mediates permeability changes.

Accelerated Lipid-Induced Atherogenesis in Galectin-3-Deficient Mice Role of Lipoxidation via Receptor-Mediated Mechanisms

Objective—Modified lipoproteins, particularly oxidized LDLs, are believed to evoke an inflammatory response which participates in all stages of atherosclerosis. Disposal of these particles is mediated through receptors which may trigger proinflammatory signaling pathways leading to vascular injury. This study was aimed at assessing the role in atherogenesis of one of these receptors, galectin-3. Methods and Results—Galectin-3–deficient and wild-type mice were fed an atherogenic diet or standard chow for 8 months. Lesion area and length were higher in galectin-3–deficient versus wild-type mice. At the level of the aortic sinus, wild-type animals showed only fatty streaks, whereas galectin-3–deficient mice developed complex lesions, associated with extensive inflammatory changes. This was indicated by the presence of T lymphocytes with activated Th1-phenotype and by more marked monocyte-macrophage infiltration, inflammatory mediator expression, vascular cell apoptosis, and proinflammatory transcription factor activation. Increased accumulation of oxidixed LDLs and lipoxidation products and upregulation of other receptors for these compounds, including the proinflammatory RAGE, were detected in galectin-3–deficient versus wild-type mice. Conclusions—These data suggest a unique protective role for galectin-3 in the uptake and effective removal of modified lipoproteins, with concurrent downregulation of proinflammatory pathways responsible for atherosclerosis initiation and progression.

Advanced lipoxidation end-products mediate lipid-induced glomerular injury: role of receptor-mediated mechanisms

Atherosclerosis and renal disease are related conditions, sharing several risk factors. This includes hyperlipidaemia, which may result in enhanced lipoprotein accumulation and chemical modification, particularly oxidation, with formation of advanced lipoxidation endproducts (ALEs). We investigated whether increased lipid peroxidation plays a major role in the pathogenesis of lipid‐induced renal disease, via receptor‐mediated mechanisms involving the scavenger and advanced glycation endproduct (AGE) receptors. Mice knocked out for galectin‐3 (Gal3−/−), an AGE receptor previously shown to protect from AGE‐induced renal injury, and the corresponding wild‐type (Gal3+/+) animals, were fed an atherogenic high‐fat diet (HFD; 15% fat, 1.25% cholesterol and 0.5% sodium cholate); mice fed a normal‐fat diet (NFD; 4% fat) served as controls. Gal3+/+ mice fed a HFD developed glomerular disease, as indicated by proteinuria, mesangial expansion and glomerular hypertrophy and sclerosis. Glomerular injury was associated with increased glomerular matrix protein expression, ALE and oxidized LDL content, oxidative stress, AGE and scavenger receptor expression and macrophage infiltration, with only modest renal/glomerular fat accumulation and changes in lipid metabolism. Fibrotic and inflammatory changes, together with accumulation of ALEs, such as 4‐hydroxy‐2‐nonenal adducts and Nε‐carboxymethyllysine, oxidative stress and expression of the receptor of AGEs (RAGE), were significantly more marked in Gal3−/− animals, whereas fat deposition and abnormalities in lipid metabolism remained modest. Thus, lipid‐induced renal damage is mainly dependent on lipid peroxidation with formation of carbonyl reactive species and ALEs, which accumulate within the kidney tissue, thus triggering receptor‐mediated pro‐inflammatory signalling pathways, as in atherogenesis. Moreover, galectin‐3 exerts a significant role in the uptake and effective removal of modified lipoproteins, with diversion of these products from RAGE‐dependent pro‐inflammatory pathways associated with downregulation of RAGE expression. Copyright

Galectin-3 ablation protects mice from diet-induced NASH: a major scavenging role for galectin-3 in liver.

BACKGROUND & AIMS Excess fatty acid oxidation and generation of reactive carbonyls with formation of advanced lipoxidation endproducts (ALEs) is involved in nonalcoholic steatohepatitis (NASH) by triggering inflammation, hepatocyte injury, and fibrosis. This study aimed at verifying the hypothesis that ablation of the ALE-receptor galectin-3 prevents experimental NASH by reducing receptor-mediated ALE clearance and downstream events. METHODS Galectin-3-deficient (Lgals3(-/-)) and wild type (Lgals3(+/+)) mice received an atherogenic diet or standard chow for 8 months. Liver tissue was analyzed for morphology, inflammation, cell and matrix turnover, lipid metabolism, ALEs, and ALE-receptors. RESULTS Steatosis was significantly less pronounced in Lgals3(-/-) than Lgals3(+/+) animals on atherogenic diet. NASH, invariably detected in Lgals3(+/+) mice, was observed, to a lower extent, only in 3/8 Lgals3(-/-) mice, showing less inflammatory, degenerative, and fibrotic phenomena than Lgals3(+/+) mice. This was associated with higher circulating ALE levels and lower tissue ALE accumulation and expression of other ALE-receptors. Up-regulation of hepatic fatty acid synthesis and oxidation, inflammatory cell infiltration, pro-inflammatory cytokines, endoplasmic reticulum stress, hepatocyte apoptosis, myofibroblast transdifferentiation, and impaired Akt phosphorylation were also significantly attenuated in Lgals3(-/-) animals. Galectin-3 silencing in liver endothelial cells resulted in reduced N(ε)-carboxymethyllysine-modified albumin uptake and ALE-receptor expression. CONCLUSIONS Galectin-3 ablation protects from diet-induced NASH by decreasing hepatic ALE accumulation, with attenuation of inflammation, hepatocyte injury, and fibrosis. It also reduced up-regulation of lipid synthesis and oxidation causing less fat deposition, oxidative stress, and possibly insulin resistance. These data suggest that galectin-3 is a major receptor involved in ALE uptake by the liver.