Metformin Prevents Endothelial Dysfunction in Endometriosis through Downregulation of ET-1 and Upregulation of eNOS.

This study aimed to evaluate if the treatment with metformin affects the morphologic structure, endothelial function, angiogenesis, inflammation and oxidation-responsive pathways in the heart of mice with surgically induced endometriosis. B6CBA/F1 mice (n = 37) were divided into four groups; Sham (S), Metformin (M), Endometriosis (E) and Metformin/Endometriosis (ME). The cross-sectional area of cardiomyocytes was assessed after Hematoxylin-Eosin staining and fibrosis after Picrosirius-Red staining. ET-1, nitric oxide synthases-iNOS and eNOS, and VEGF and VEGFR-2 were detected by immunofluorescence. Semi-quantification of ET-1, eNOS, VEGF, NF-kB, Ikßα and KEAP-1 was performed by Western blotting. MIR199a, MIR16-1, MIR18a, MIR20a, MIR155, MIR200a, MIR342, MIR24-1 and MIR320a were quantified by Real-Time qPCR. The interaction of endometriosis and metformin effects was assessed by a two-way ANOVA test. Compared with the other groups, M-treated mice presented a higher cross-sectional area of cardiomyocytes. Heart fibrosis increased with endometriosis. Treatment of endometriosis with metformin in the ME group downregulates ET-1 and upregulates eNOS expression comparatively with the E group. However, metformin failed to mitigate NF-kB expression significantly incremented by endometriosis. The expression of MIR199a, MIR16-1 and MIR18a decreased with endometriosis, whereas MIR20a showed an equivalent trend, altogether reducing cardioprotection. In summary, metformin diminished endometriosis-associated endothelial dysfunction but did not mitigate the increase in NF-kB expression and cardiac fibrosis in mice with endometriosis.

Transthyretin participates in beta-amyloid transport from the brain to the liver--involvement of the low-density lipoprotein receptor-related protein 1?

Transthyretin (TTR) binds Aß peptide, preventing its deposition and toxicity. TTR is decreased in Alzheimer's disease (AD) patients. Additionally, AD transgenic mice with only one copy of the TTR gene show increased brain and plasma Aß levels when compared to AD mice with both copies of the gene, suggesting TTR involvement in brain Aß efflux and/or peripheral clearance. Here we showed that TTR promotes Aß internalization and efflux in a human cerebral microvascular endothelial cell line, hCMEC/D3. TTR also stimulated brain-to-blood but not blood-to-brain Aß permeability in hCMEC/D3, suggesting that TTR interacts directly with Aß at the blood-brain-barrier. We also observed that TTR crosses the monolayer of cells only in the brain-to-blood direction, as confirmed by in vivo studies, suggesting that TTR can transport Aß from, but not into the brain. Furthermore, TTR increased Aß internalization by SAHep cells and by primary hepatocytes from TTR+/+ mice when compared to TTR-/- animals. We propose that TTR-mediated Aß clearance is through LRP1, as lower receptor expression was found in brains and livers of TTR-/- mice and in cells incubated without TTR. Our results suggest that TTR acts as a carrier of Aß at the blood-brain-barrier and liver, using LRP1.

Transthyretin stabilization by iododiflunisal promotes amyloid-ß peptide clearance, decreases its deposition, and ameliorates cognitive deficits in an Alzheimer's disease mouse model.

Alzheimer's disease (AD) is the most common form of dementia and now represents 50-70% of total dementia cases. Over the last two decades, transthyretin (TTR) has been associated with AD and, very recently, a novel concept of TTR stability has been established in vitro as a key factor in TTR/amyloid-ß (Aß) interaction. Small compounds, TTR stabilizers (usually non-steroid anti-inflammatory drugs), bind to the thyroxine (T4) central binding channel, increasing TTR tetrameric stability and TTR/Aß interaction. In this work, we evaluated in vivo the effects of one of the TTR stabilizers identified as improving TTR/Aß interaction, iododiflunisal (IDIF), in Aß deposition and other AD features, using AßPPswe/PS1A246E transgenic mice, either carrying two or just one copy of the TTR gene (AD/TTR+/+ or AD/TTR+/-, respectively), available and characterized in our laboratory. The results showed that IDIF administered orally bound TTR in plasma and stabilized the protein, as assessed by T4 displacement assays, and was able to enter the brain as revealed by mass spectrometry analysis of cerebrospinal fluid. TTR levels, both in plasma and cerebrospinal fluid, were not altered. In AD/TTR+/- mice, IDIF administration resulted not only in decreased brain Aß levels and deposition but also in improved cognitive function associated with the AD-like neuropathology in this mouse model, although no improvements were detectable in the AD/TTR+/+ animals. Further, in AD/TTR+/- mice, Aß levels were reduced in plasma suggesting TTR promoted Aß clearance from the brain and from the periphery. Taken together, these results strengthen the importance of TTR stability in the design of therapeutic drugs, highlighting the capacity of IDIF to be used in AD treatment to prevent and to slow the progression of the disease.

AMP-activated protein kinase phosphorylates cardiac troponin I and alters contractility of murine ventricular myocytes.

RATIONALE: AMP-activated protein kinase (AMPK) is an important regulator of energy balance and signaling in the heart. Mutations affecting the regulatory γ2 subunit have been shown to cause an essentially cardiac-restricted phenotype of hypertrophy and conduction disease, suggesting a specific role for this subunit in the heart. OBJECTIVE: The γ isoforms are highly conserved at their C-termini but have unique N-terminal sequences, and we hypothesized that the N-terminus of γ2 may be involved in conferring substrate specificity or in determining intracellular localization. METHODS AND RESULTS: A yeast 2-hybrid screen of a human heart cDNA library using the N-terminal 273 residues of γ2 as bait identified cardiac troponin I (cTnI) as a putative interactor. In vitro studies showed that cTnI is a good AMPK substrate and that Ser150 is the principal residue phosphorylated. Furthermore, on AMPK activation during ischemia, Ser150 is phosphorylated in whole hearts. Using phosphomimics, measurements of actomyosin ATPase in vitro and force generation in demembraneated trabeculae showed that modification at Ser150 resulted in increased Ca(2+) sensitivity of contractile regulation. Treatment of cardiomyocytes with the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) resulted in increased myocyte contractility without changing the amplitude of Ca(2+) transient and prolonged relaxation despite shortening the time constant of Ca(2+) transient decay (tau). Compound C prevented the effect of AICAR on myocyte function. These results suggest that AMPK activation increases myocyte contraction and prolongs relaxation by increasing myofilament Ca(2+) sensitivity. CONCLUSIONS: We conclude that cTnI phosphorylation by AMPK may represent a novel mechanism of regulation of cardiac function.

Transthyretin: roles in the nervous system beyond thyroxine and retinol transport.

Transthyretin (TTR) is a plasma- and cerebrospinal fluid-circulating protein. Besides the primordially attributed systemic role as a transporter molecule of thyroxine (T4) and retinol (through the binding to retinol-binding protein [RBP]), TTR has been recognized as a protein with important functions in several aspects of the nervous system physiology. TTR has been shown to play an important role in behavior, cognition, amidated neuropeptide processing and nerve regeneration. Furthermore, it has been proposed that TTR is neuroprotective in Alzheimer's disease and cerebral ischemia. Mutations in TTR are a well-known cause of familial amyloidotic polyneuropathy, an autosomal dominant neurodegenerative disorder characterized by systemic deposition of TTR amyloid fibrils, particularly in the peripheral nervous system. The purpose of this review is to highlight the roles of TTR in the nervous system, beyond its systemic role as a transporter molecule of T4 and RBP-retinol.

Gender-dependent transthyretin modulation of brain amyloid-ß levels: evidence from a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disorder affecting tens of millions of people worldwide, with women being at greater risk of developing the disease. A growing body of evidence suggests transthyretin (TTR) as an important modulator of AD pathogenesis. Aiming at providing further insight into the potential neuroprotective role of TTR and gender differences in AD, we crossed transgenic AßPPswe/PS1A246E mice with TTR-null mice and investigated both male and female AßPPswe/PS1A246E/TTR+/+, AßPPswe/PS1A246E/TTR+/-, and AßPPswe/PS1A246E/TTR-/- animals for brain amyloid-ß (Aß) levels and deposition. The levels of circulating TTR between non-transgenic and AD mice were evaluated. Decreased levels of circulating TTR in AD mice as compared to non-transgenic littermates were observed in early stages of AD-like neuropathology, but not at later stages where an opposite relationship was found. Elevated brain levels of Aß42 were observed in AßPPswe/PS1A246E/TTR+/- female mice as compared to AßPPswe/PS1A246E/TTR+/+ female littermates; no significant differences were found among males of different TTR genotypes. We subsequently quantified the brain levels of testosterone and 17ß-estradiol in these animals and verified that AßPPswe/PS1A246E/TTR+/- female mice present reduced brain levels of both hormones as compared to AßPPswe/PS1A246E/TTR+/+ females; no significant differences were detected among males of different TTR genotypes. Our results provide evidence for a gender-associated modulation of brain Aß levels and brain sex steroid hormones by TTR, and suggest that reduced levels of brain testosterone and 17ß-estradiol in female mice with TTR genetic reduction might underlie their increased AD-like neuropathology.

Mutation analysis of AMP-activated protein kinase subunits in inherited cardiomyopathies: implications for kinase function and disease pathogenesis.

Familial hypertrophic cardiomyopathy (HCM) has been defined as a disease of the cardiac sarcomere, although sarcomeric protein mutations are not found in one third of cases. We have recently shown that HCM associated with Wolff-Parkinson-White syndrome (WPW) and conduction disease can be caused by mutations in PRKAG2, which encodes the gamma2 subunit of AMPK, an enzyme central to cellular energy homeostasis. AMPK is a heterotrimer composed of one catalytic subunit (alpha) and two regulatory subunits (beta and gamma). Seven known genes encode the subunit isoforms (alpha1, alpha2, beta1, beta2, gamma1, gamma2, gamma3) and all are expressed in the heart. To better understand the role of AMPK mutations in HCM/WPW and other inherited cardiomyophathies, all 7 subunit genes were screened for mutations in a panel of probands: 3 with HCM/WPW, 4 with DCM/WPW, 38 with HCM alone (in whom contractile protein mutations had not been found) and 13 with DCM alone. In total, 73 amplimers were screened in the 58 probands and a number of polymorphisms, including non-conservative substitutions, were identified. However, no further disease-causing mutations were found in any AMPK subunit gene. These results indicate that HCM with WPW is a distinct, but genetically heterogeneous, condition caused by mutations in PRKAG2 and in an unknown gene or genes, not involved in the AMPK complex. Mutations in PRKAG2 appear to specifically cause HCM with WPW and conduction disease, and not other inherited cardiomyopathies. As deleterious alleles were not found in other AMPK subunit isoforms, the mutations affecting PRKAG2 are likely to confer a specific alteration of AMPK function of particular importance in the myocardium.