A New Perspective in Utilizing MMP-9 as a Therapeutic Target for Alzheimer's Disease and Type 2 Diabetes Mellitus.

Matrix metalloprotease 9 (MMP-9) is a 92 kDa type IV collagenase and a member of the family of endopeptidases. MMP-9 is involved in the degradation of extracellular matrix components, tissue remodeling, cellular receptor stripping, and processing of various signaling molecules. In the CNS, the effects of MMP-9 are quite complex, since it exerts beneficial effects including neurogenesis, angiogenesis, myelogenesis, axonal growth, and inhibition of apoptosis, or destructive effects including apoptosis, blood-brain barrier disorder, and demyelination. Likewise, in the periphery, physiological events, as the involvement of MMP-9 in angiogenesis, for instance in wound healing, can be turned into pathological, such as in tumor metastasis, depending on the state of the organism. Alzheimer's disease is a neurodegenerative disorder, characterized by amyloid accumulation and deposition in the brain. Amyloidogenesis, however, also occurs in diseases of the periphery, such as type II diabetes mellitus, where an analogous type of amyloid, is deposited in the pancreas. Interestingly, both diseases exhibit similar pathology and disease progression, with insulin resistance being a major common denominator. Hence, combinatorial strategies searching new or existing molecules to apply for therapeutic use for both diseases are gaining momentum. MMP-9 is extensively studied due to its association with a variety of physiological and pathological processes. Consequently, meticulous design could render MMP-9 into a potential therapeutic target for Alzheimer's disease and type 2 diabetes mellitus; two seemingly unrelated diseases.

A Review on Platelet Activating Factor Inhibitors: Could a New Class of Potent Metal-Based Anti-Inflammatory Drugs Induce Anticancer Properties?

In this minireview, we refer to recent results as far as the Platelet Activating Factor (PAF) inhibitors are concerned. At first, results of organic compounds (natural and synthetic ones and specific and nonspecific) as inhibitors of PAF are reported. Emphasis is given on recent results about a new class of the so-called metal-based inhibitors of PAF. A small library of 30 metal complexes has been thus created; their anti-inflammatory activity has been further evaluated owing to their inhibitory effect against PAF in washed rabbit platelets (WRPs). In addition, emphasis has also been placed on the identification of preliminary structure-activity relationships for the different classes of metal-based inhibitors.

Overexpression of matrix metalloproteinase-9 (MMP-9) rescues insulin-mediated impairment in the 5XFAD model of Alzheimer's disease.

A hallmark of Alzheimer's disease (AD) is the accumulation of oligomeric amyloid-ß (Aß) peptide, which may be primarily responsible for neuronal dysfunction. Insulin signaling provides a defense mechanism against oligomer-induced neuronal loss. We previously described the neuroprotective role of matrix metalloproteinase 9 (MMP-9) in decreasing the formation of Aß oligomers. In the present study, we examined the role of MMP-9 on the insulin survival pathway in primary hippocampal cultures and hippocampal cell extracts from 3 month-old wild type, AD (5XFAD), MMP-9-overexpressing (TgMMP-9), and double transgenic mice (5XFAD/TgMMP-9). The data demonstrate that the insulin pathway was compromised in samples from 5XFAD mice, when compared to the wild type and TgMMP-9. This was due to enhanced phosphorylation of IRS1 at Serine 636 (pIRS1-Ser636), which renders IRS1 inactive and prevents insulin-mediated signaling. In 5XFAD/TgMMP-9 samples, the insulin survival pathway was rescued through enhanced activation by phosphorylation of IRS1 at Tyrosine 465 (pIRS1-Tyr465), downstream increased phosphorylation of Akt and GSK-3ß, and decreased phosphorylation of JNK kinase. Oligomeric Aß levels decreased and BDNF levels increased in 5XFAD/TgMMP-9 mice, compared to 5XFAD mice. Our findings indicate that overexpression of MMP-9 rescued insulin survival signaling in vitro and in early stages in the 5XFAD model of AD.

The ability of apolipoprotein E fragments to promote intraneuronal accumulation of amyloid beta peptide 42 is both isoform and size-specific.

The apolipoprotein (apo) E4 isoform is the strongest risk factor for late-onset Alzheimer's disease (AD). ApoE4 is more susceptible to proteolysis than apoE2 and apoE3 isoforms and carboxyl-terminal truncated apoE4 forms have been found in AD patients' brain. We have previously shown that a specific apoE4 fragment, apoE4-165, promotes amyloid-peptide beta 42 (Aß42) accumulation in human neuroblastoma SK-N-SH cells and increased intracellular reactive oxygen species formation, two events considered to occur early in AD pathogenesis. Here, we show that these effects are allele-dependent and absolutely require the apoE4 background. Furthermore, the exact length of the fragment is critical since longer or shorter length carboxyl-terminal truncated apoE4 forms do not elicit the same effects. Structural and thermodynamic analyses showed that apoE4-165 has a compact structure, in contrast to other carboxyl-terminal truncated apoE4 forms that are instead destabilized. Compared however to other allelic backgrounds, apoE4-165 is structurally distinct and less thermodynamically stable suggesting that the combination of a well-folded structure with structural plasticity is a unique characteristic of this fragment. Overall, our findings suggest that the ability of apoE fragments to promote Aß42 intraneuronal accumulation is specific for both the apoE4 isoform and the particular structural and thermodynamic properties of the fragment.

High Glucose Impairs Insulin Signaling in the Glomerulus: An In Vitro and Ex Vivo Approach.

OBJECTIVE: Chronic hyperglycaemia, as seen in type II diabetes, results in both morphological and functional impairments of podocytes in the kidney. We investigated the effects of high glucose (HG) on the insulin signaling pathway, focusing on cell survival and apoptotic markers, in immortalized human glomerular cells (HGEC; podocytes) and isolated glomeruli from healthy rats. METHODS AND FINDINGS: HGEC and isolated glomeruli were cultured for various time intervals under HG concentrations in the presence or absence of insulin. Our findings indicated that exposure of HGEC to HG led to downregulation of all insulin signaling markers tested (IR, p-IR, IRS-1, p-Akt, p-Fox01,03), as well as to increased sensitivity to apoptosis (as seen by increased PARP cleavage, Casp3 activation and DNA fragmentation). Short insulin pulse caused upregulation of insulin signaling markers (IR, p-IR, p-Akt, p-Fox01,03) in a greater extent in normoglycaemic cells compared to hyperglycaemic cells and for the case of p-Akt, in a PI3K-dependent manner. IRS-1 phosphorylation of HG-treated podocytes was negatively regulated, favoring serine versus tyrosine residues. Prolonged insulin treatment caused a significant decrease of IR levels, while alterations in glucose concentrations for various time intervals demonstrated changes of IR, p-IR and p-Akt levels, suggesting that the IR signaling pathway is regulated by glucose levels. Finally, HG exerted similar effects in isolated glomeruli. CONCLUSIONS: These results suggest that HG compromises the insulin signaling pathway in the glomerulus, promoting a proapoptotic environment, with a possible critical step for this malfunction lying at the level of IRS-1 phosphorylation; thus we herein demonstrate glomerular insulin signaling as another target for investigation for the prevention and/ or treatment of diabetic nephropathy.

Glucose-induced gradual phenotypic modulation of cultured human glomerular epithelial cells may be independent of Wilms' tumor 1 (WT1).

BACKGROUND: Renal podocytes form the main filtration barrier possessing a unique phenotype maintained by proteins including podocalyxin and nephrin, the expression of which is suppressed in pathological conditions. We used an in vitro model of human glomerular epithelial cells (HGEC) to investigate the role of high glucose in dysregulating the podocytic epithelial phenotype and determined the time needed for this change to occur. RESULTS: In our in vitro podocyte system changes indicating podocyte dedifferentiation in the prolonged presence of high glucose included loss of podocalyxin, nephrin and CD10/CALLA concomitant with upregulation of mesenchymal vimentin. Our study demonstrates for the first time that podocyte-specific markers undergo changes of expression at different time intervals, since glucose-mediated podocalyxin downregulation is a progressive process that precedes downregulation of nephrin expression. Finally we demonstrate that high glucose permanently impaired WT1 binding to the podocalyxin gene promoter region but did not affect WT1 binding on the nephrin gene promoter region. CONCLUSION: The presence of high glucose induced a phenotypic conversion of podocytes resembling partial dedifferentiation. Our study demonstrates that dysregulation of the normal podocytic phenotype is an event differentially affecting the expression of function-specific podocytic markers, exhibiting downregulation of the epithelial marker CD10/CALLA and PC first, followed by stably downregulated nephrin. Furthermore, it is herein suggested that WT1 may not be directly involved with upregulation of previously reduced PC and nephrin expression.

Oleuropein, an anti-oxidant polyphenol constituent of olive promotes α-secretase cleavage of the amyloid precursor protein (AßPP).

Over the past decade, intense focus has been dedicated on investigating processes involved in the proteolysis of amyloid precursor protein (AßPP) and ß-amyloid (Aß) peptide metabolism, as possible targets for Alzheimer's disease (AD) therapy. To this goal, considerable research has been targeted on potential therapeutic use of compounds promoting non-amyloidogenic processing of AßPP. One of these compounds, oleuropein, a polyphenol constituent of extra virgin olive oil exhibiting a wide range of pharmacological properties, was shown to interact non-covalently with Aß, an interaction that might be related to a potential protective role of oleuropein against Aß aggregation. In the present study, it was demonstrated that oleuropein treatment of HEK293 cells stably transfected with the isoform 695 of human AßPP (APP695) leads to markedly elevated levels of sAPPα and to significant reduction of Aß oligomers. These effects were associated with increased activity of matrix metalloproteinase 9 (MMP-9), whereas no significant alterations in the expression of secretases TACE, ADAM-10 or BACE-1 were observed. Similar results were obtained using the human neuroblastoma cell line SK-N-SH. The experimental data reveal an anti-amyloidogenic effect of oleuropein and suggest a possible protective role for oleuropein against AD, extending the spectrum of beneficial properties of this naturally occurring polyphenol.

Matrix metalloproteinase-9 participates in NGF-induced α-secretase cleavage of amyloid-ß protein precursor in PC12 cells.

Amyloid-ß protein precursor (AßPP) is a ubiquitously expressed glycoprotein, which under physiological conditions can be cleaved following two alternative routes; the non-amyloidogenic and the amyloidogenic pathway. Shift of AßPP processing in favor of the amyloidogenic pathway is a key event in the pathogenesis of Alzheimer's disease (AD). Among the factors that regulate AßPP processing, nerve growth factor (NGF) appears to play an important role; abnormal NGF signaling has been implicated in the onset of AD. In the present study, we used PC12 cells to study the effects of NGF on AßPP processing and provide evidence that NGF, through binding to its high affinity receptor, TrkA moderately down-regulates the expression of the ß-secretase ß-site AßPP cleaving enzyme-1 and, most importantly, upregulates the expression of two enzymes with α-secretase activity, a disintegrin and metalloprotease-17 and to a greater extent matrix metalloproteinase-9 (MMP9) in a phosphoinositide kinase-3 dependent manner. Finally, we demonstrate that MMP9 actively participates in NGF-induced α-secretase cleavage of AßPP, thus it contributes to the shift of AßPP processing towards the non-amyloidogenic pathway precluding the formation of neurotoxic Aß peptides.

TIMP-1 interaction with αvß3 integrin confers resistance to human osteosarcoma cell line MG-63 against TNF-α-induced apoptosis.

Tumor necrosis factor-α (TNF-α) is a pleiotropic cytokine affecting diverse cellular responses. TNF-α is cytotoxic in many systems, but it can also act as an anti-apoptotic signal to promote cell survival pathways activated through integrins and extracellular matrix components. This is particularly evident in cancer cells. To unravel the basis of resistance to TNF-α-induced apoptosis, human osteosarcoma MG-63 cell line was used. Our data showed that resistance to apoptosis was accompanied by high levels of TIMP-1 expression in part mediated by NF-κB activation, whereas under apoptotic conditions, in the presence of cycloheximide (CHX), TIMP-1 and αvß3 integrin protein levels were significantly reduced. Silencing TIMP-1 using siRNA led to increased apoptosis following treatment with TNF-α, whereas exogenously-added recombinant TIMP-1 reduced the extent of apoptosis. Immunoprecipitation and confocal microscopy experiments demonstrated that TIMP-1 interacted with αvß3 integrins. The biological role of this interaction was revealed by the use of echistatin, an antagonist of αvß3 integrin. In the presence of echistatin, decreased protection against apoptosis by recombinant TIMP-1 was observed.

An apolipoprotein E4 fragment can promote intracellular accumulation of amyloid peptide beta 42.

Apolipoprotein E (apoE) plays a crucial role in lipid transport in circulation and the brain. The apoE4 isoform is a major risk factor for Alzheimer's disease (AD). ApoE4 is more susceptible to proteolysis than other apoE isoforms and apoE4 fragments have been found in brains of AD patients. These apoE4 fragments have been hypothesized to be involved in the pathogenesis of AD, although the mechanism is not clear. In this study we examined the effect of lipid-free apoE4 on amyloid precursor protein processing and 40-amino-acid Aß variant and 42-amino-acid Aß variant levels in human neuroblastoma SK-N-SH cells. We discovered that a specific apoE4 fragment, apoE4[Δ(166-299)], can promote the cellular uptake of extracellular 40-amino-acid Aß variant and 42-amino-acid Aß variant either generated after amyloid precursor protein transfection or added exogenously. A longer length fragment, apoE4[Δ(186-299)], or full-length apoE4 failed to elicit this effect. ApoE4[Δ(166-299)] effected a 20% reduction of cellular sphingomyelin levels, as well as changes in cellular membrane micro-fluidity. Following uptake, approximately 50% of 42-amino-acid Aß variant remained within the cell for at least 24 h, and led to increased formation of reactive oxygen species. Overall, our findings suggest a direct link between two early events in the pathogenesis of AD, apoE4 proteolysis and intraneuronal presence of amyloid beta peptide.

High glucose induces suppression of insulin signalling and apoptosis via upregulation of endogenous IL-1beta and suppressor of cytokine signalling-1 in mouse pancreatic beta cells.

Chronic hyperglycemia and inflammatory cytokines disrupt and/or attenuate signal transduction pathways that promote normal beta-cell survival, leading to the destruction of endocrine pancreas in type 2 diabetes. There is convincing evidence that autocrine insulin signalling exerts protective anti-apoptotic effects on beta cells. Suppressors of cytokine signalling (SOCS) were induced by several cytokines and inhibit insulin-initiated signal transduction. The aim of this study was to investigate whether high glucose can influence endogenous interleukin-1beta (IL-1beta) and SOCS expression thus affecting insulin signalling and survival in insulin-producing mouse pancreatic beta cells (betaTC-6). Results showed that prolonged exposure of betaTC-6 cells to increased glucose concentrations resulted in significant inhibition of insulin-induced tyrosine phosphorylation of the insulin receptor (IR), and insulin receptor substrate-2 (IRS-2) as well as PI3-kinase activation. These changes were accompanied by impaired activation of the anti-apoptotic signalling protein Akt and annulment of Akt-mediated suppression of the Forkhead family of transcription factors (FoxO) activation. Glucose-induced attenuation of IRS-2/Akt-mediated signalling was associated with increased IL-1beta expression. Enhanced endogenous IL-1beta specifically induced mRNA and protein expression of SOCS-1 in betaTC-6 cells. Inhibition of SOCS-1 expression by SOCS-1-specific small interfering RNA restored IRS-2/PI3K-mediated Akt phosphorylation suppressed by high glucose. The upregulation of endogenous cytokine signalling and FoxO activation were accompanied by enhanced caspase-3 activation and increased susceptibility of cells to apoptosis. These results indicated that glucose-induced endogenous IL-1beta expression increased betaTC-6 cells apoptosis by inhibiting, at least in part, IRS-2/Akt-mediated signalling through SOCS-1 upregulation.

Impaired transcription factor interplay in addition to advanced glycation end products suppress podocalyxin expression in high glucose-treated human podocytes.

Podocalyxin represents a Wilms' tumor suppressor protein (WT1)-regulated differentiation marker for glomerular epithelium. We provide evidence concerning mechanisms involved in the regulation of podocalyxin expression following long-term exposure to increased (25 mM) glucose levels. Prolonged culture of conditionally immortalized human podocytes in 25 mM glucose induced suppression of podocalyxin expression both at the protein and mRNA levels, whereas WT1 protein levels remained unaltered. WT1 interacted with another transcription factor, CRE-binding protein (CBP). This association was decreased by 40% in the presence of 25 mM glucose. Chromatin immunoprecipitation assays on chromatin from podocytes cultured in 25 mM glucose revealed reduced WT1 binding to podocalyxin promoter sequences, probably resulting from impaired WT1-CBP interactions. We explored the possible role of glucose-induced adducts (advanced glycation end products; AGEs) in impairing interactions between WT1 and CBP, with the use of aminoguanindine, an inhibitor of AGE formation. Podocytes were cultured in the simultaneous presence of 20 mM aminoguanidine and 25 mM glucose, and podocalyxin protein levels were examined. Aminoguanidine effectively prevented downregulation of podocalyxin protein levels but could not restore podocalyxin levels once expression was suppressed. Thus increased glucose apparently impaired the ability of WT1 to initiate transcription in part by decreased association of WT1 with CBP. Administration of aminoguanidine concomitant with increasing glucose levels in our in vitro model system protected from glucose-induced "silencing" of the podocalyxin gene, suggesting that AGEs play an important role in suppressing its expression in diabetic conditions.

Selective binding of integrins from different renal cell types to the NC1 domain of alpha3 and alpha1 chains of type IV collagen.

BACKGROUND: Cells interact with type IV collagen (Col IV) via integrins through the triple-helical and NC1 domains. We examined interactions of human glomerular and proximal tubular epithelial cells with recombinant alpha1 and alpha3 NC1 chains of Col IV, to explore the ability of different cell types to interact with Col IV of different trimer composition. METHODS: Interactions of TSV-40-immortalized human glomerular epithelial cells (HGECs), HPV-16-immortalized human proximal tubular epithelial (HK-2) cells and primary human mesangial cells (MES) with recombinant alpha1 and alpha3 NC1 chains of Col IV were examined by affinity chromatography and solid-phase binding assays. The expression of integrin-regulated metalloproteinases was examined by zymography. RESULTS: HGECs bound to both alpha3 and alpha1(IV)NC1, albeit there was preferential binding to alpha3(IV)NC1, through the alpha3beta1 and alpha2beta1 integrin receptors; HK-2 cells and MES bound almost exclusively to alpha1(IV)NC1 via the alpha3beta1/alphavbeta3 and alpha1beta1/alpha2beta1 receptors, respectively. It was demonstrated that the expression of MMP-2 and MMP-9 by HGECs was down-regulated in the presence of alpha3(IV)NC1. CONCLUSIONS: The observed data indicate that the isoform NC1 chains of Col IV serve for selective, integrin-mediated cell binding which probably triggers different signaling mechanisms, resulting in the activation of specific transcription factors and the modulation of gene expression.

Enhanced podocalyxin expression alters the structure of podocyte basal surface.

Glomerular basement membrane (GBM) and podocalyxin are essential for podocyte morphology. We provide evidence of functional interconnections between basement membrane components (collagen IV and laminin), the expression of podocalyxin and the morphology of human glomerular epithelial cells (podocytes). We demonstrated that GBM and laminin, but not collagen IV, up-regulated the expression of podocalyxin. Scanning electron microscopy revealed that laminin induced a modified morphology of podocytes with process formation, which was more extensive in the presence of GBM. Under high magnification, podocytes appeared ruffled. Using transmission electron microscopy we observed that raised areas occurred in the basal cell surface. Furthermore, the presence of anti-podocalyxin antibody increased the extent of adhesion and spreading of podocytes to both collagen IV and laminin, thus podocalyxin apparently inhibits cell-matrix interactions. We also performed adhesion and spreading assays on podocytes grown under increased glucose concentration (25 mM). Under these conditions, the expression of podocalyxin was almost totally suppressed. The cells adhered and spread to basement membrane components but there was no increase in the extent of adhesion and spreading in the presence of anti-podocalyxin antibody, or ruffling of the cell edges. Additionally, in podocytes expressing podocalyxin, the presence of anti-podocalyxin antibody partially reversed the inhibition of adhesion to collagen IV provoked by anti-beta1 integrin antibody, thus podocalyxin should compete with beta1-related cell adhesion. We suggest that the observed podocalyxin-mediated inhibition of binding to the matrix could be in part responsible for the specialized conformation of the basal surface of podocytes.

Glucose-induced changes in integrins and matrix-related functions in cultured human glomerular epithelial cells.

In cultured human glomerular epithelial cells (HGEC), 25 mM glucose resulted in decreased expression of alpha(3)-, alpha(2)-, and beta(1)-integrins and increased expression of alpha(5)- and alpha(v)beta(3)-integrins. This change was accompanied by decreased binding of HGEC to type IV collagen. In the presence of normal (5 mM) glucose concentration, cell binding to type IV collagen was primarily mediated by alpha(2)beta(1)- and alpha(5)beta(1)-integrins, as indicated by experiments in which cell adhesion to type IV collagen was competed by specific anti-integrin monoclonal antibodies. In the presence of high (25 mM) glucose, the upregulated alpha(5)- and alpha(v)beta(3)-integrins were mainly involved in cell binding to type IV collagen. Furthermore, high glucose decreased expression of matrix metalloproteinase-2 (MMP-2), a collagenase regulated in part by alpha(3)beta(1)-integrin, as suggested by the use of ligand-mimicking antibodies against these integrins, which resulted in release of increased amounts of MMP-2 in the culture medium. Finally, tissue inhibitor of metalloproteinase-2, the specific inhibitor of MMP-2, was upregulated in high glucose and could contribute to matrix accumulation. These changes could help explain basement membrane thickening in diabetes.

Effects of collagen IV on neuroblastoma cell matrix-related functions.

Integrin-mediated interactions with collagen IV and its domains were examined in a human neuroblastoma cell line (SK-N-SH). By adhesion assays we demonstrated that neuroblastoma cells bound to solid-phase intact collagen IV and synthetic cell-binding peptide HEP-III, derived from the collagenous part of the molecule, but not to the main noncollagenous NC1 domain or to the synthetic cell-binding peptide HEP-I, derived from this domain. Monoclonal antibodies against beta1, alpha3, and alpha(v)beta3 integrins resulted in inhibition of cell binding to collagenous substrates by 95, 30, and 35%, respectively. By flow cytometry and immunoblotting it was shown that culture of SK-N-SH cells on collagen IV resulted in alteration in the expression of major neuroblastoma cell integrins. Binding to collagen IV induced the expression and activation of matrix metalloproteinases A and B (MMP-2, MMP-9), with a concomitant increase at the protein level of tissue-specific inhibitors of metalloproteinases (TIMP-1, TIMP-2). Finally, the expression of MMP-2 was significantly up-regulated by anti-alpha3beta1 antibodies, whereas ligation of anti-alpha(v)beta3 antibodies resulted in a modest down-regulation of MMP-2. Our results indicate that the presence of collagen IV modulates the expression of integrins, which are used for binding to this glycoprotein, and MMP-2 secreted by SK-N-SH cells.