Absence of Myocardial Thyroid Hormone Inactivating Deiodinase Results in Restrictive Cardiomyopathy in Mice

Cardiac injury induces myocardial expression of the thyroid hormone inactivating type 3 deiodinase (D3), which in turn dampens local thyroid hormone signaling. Here, we show that the D3 gene (Dio3) is a tissue-specific imprinted gene in the heart, and thus, heterozygous D3 knockout (HtzD3KO) mice constitute a model of cardiac D3 inactivation in an otherwise systemically euthyroid animal. HtzD3KO newborns have normal hearts but later develop restrictive cardiomyopathy due to cardiac-specific increase in thyroid hormone signaling, including myocardial fibrosis, impaired myocardial contractility, and diastolic dysfunction. In wild-type littermates, treatment with isoproterenol-induced myocardial D3 activity and an increase in the left ventricular volumes, typical of cardiac remodeling and dilatation. Remarkably, isoproterenol-treated HtzD3KO mice experienced a further decrease in left ventricular volumes with worsening of the diastolic dysfunction and the restrictive cardiomyopathy, resulting in congestive heart failure and increased mortality. These findings reveal crucial roles for Dio3 in heart function and remodeling, which may have pathophysiologic implications for human restrictive cardiomyopathy

Modulators of Tumor Angiogenesis: Insights into the Role of Galectin-3 and IL-17 Signaling

Angiogenesis is a pivotal point in tumor progression driven by firmly orchestrated process of forming the new blood vessels relying on the complex signaling network. Here, the pleiotropic functions of Galectin-3 and IL-17 in tumor progression have been overviewed through their impacts on angiogenesis. As a key player in tumor microenvironment, Galectin-3 orchestrates practically all critical events during angiogenic cascade through interaction with various ligands and their downstream signaling pathways. Galectin-3 shapes chronic inflammatory tumor microenvironment that is closely related to angiogenesis by sharing common signaling cascades and molecules. In chronic inflammatory makeup of tumor microenvironment, IL-17 contributes to tumorigenesis and progression via promoting critical events such as angiogenesis and creation of immunosuppressive milieu. VEGF, as the master regulator of tumor angiogenesis, is the main target of Galectin-3 and IL-17 action. The better understanding of Galectin-3 and IL-17 in tumor biology will undoubtedly contribute to controlling tumor progression. Therefore, as important modulators of tumor angiogenesis, Galectin-3 and IL-17 may be perceived as the potential therapeutic targets in tumor including anti-angiogenic therapy

c-Myc is essential to prevent endothelial pro-inflammatory senescent phenotype.

The proto-oncogene c-Myc is vital for vascular development and promotes tumor angiogenesis, but the mechanisms by which it controls blood vessel growth remain unclear. In the present work we investigated the effects of c-Myc knockdown in endothelial cell functions essential for angiogenesis to define its role in the vasculature. We provide the first evidence that reduction in c-Myc expression in endothelial cells leads to a pro-inflammatory senescent phenotype, features typically observed during vascular aging and pathologies associated with endothelial dysfunction. c-Myc knockdown in human umbilical vein endothelial cells using lentivirus expressing specific anti-c-Myc shRNA reduced proliferation and tube formation. These functional defects were associated with morphological changes, increase in senescence-associated-β-galactosidase activity, upregulation of cell cycle inhibitors and accumulation of c-Myc-deficient cells in G1-phase, indicating that c-Myc knockdown in endothelial cells induces senescence. Gene expression analysis of c-Myc-deficient endothelial cells showed that senescent phenotype was accompanied by significant upregulation of growth factors, adhesion molecules, extracellular-matrix components and remodeling proteins, and a cluster of pro-inflammatory mediators, which include Angptl4, Cxcl12, Mdk, Tgfb2 and Tnfsf15. At the peak of expression of these cytokines, transcription factors known to be involved in growth control (E2f1, Id1 and Myb) were downregulated, while those involved in inflammatory responses (RelB, Stat1, Stat2 and Stat4) were upregulated. Our results demonstrate a novel role for c-Myc in the prevention of vascular pro-inflammatory phenotype, supporting an important physiological function as a central regulator of inflammation and endothelial dysfunction

Endoplasmic Reticulum Stress Decreases Intracellular Thyroid Hormone Activation via an eIF2a-Mediated Decrease in Type 2 Deiodinase Synthesis

Abstract Cells respond rapidly to endoplasmic reticulum (ER) stress by blocking protein translation, increasing protein folding capacity, and accelerating degradation of unfolded proteins via ubiquitination and ER-associated degradation pathways. The ER resident type 2 deiodinase (D2) is normally ubiquitinated and degraded in the proteasome, a pathway that is accelerated by enzyme catalysis of T4 to T3. To test whether D2 is normally processed through ER-associated degradation, ER stress was induced in cells that endogenously express D2 by exposure to thapsigargin or tunicamycin. In all cell models, D2 activity was rapidly lost, to as low as of 30% of control activity, without affecting D2 mRNA levels; loss of about 40% of D2 activity and protein was also seen in human embryonic kidney 293 cells transiently expressing D2. In primary human airway cells with ER stress resulting from cystic fibrosis, D2 activity was absent. The rapid ER stress-induced loss of D2 resulted in decreased intracellular D2-mediated T3 production. ER stress-induced loss of D2 was prevented in the absence of T4, by blocking the proteasome with MG-132 or by treatment with chemical chaperones. Notably, ER stress did not alter D2 activity half-life but rather decreased D2 synthesis as assessed by induction of D2 mRNA and by [35S]methionine labeling. Remarkably, ER-stress-induced loss in D2 activity is prevented in cells transiently expressing an inactive eukaryotic initiation factor 2, indicating that this pathway mediates the loss of D2 activity. In conclusion, D2 is selectively lost during ER stress due to an eukaryotic initiation factor 2-mediated decrease in D2 synthesis and sustained proteasomal degradation. This explains the lack of D2 activity in primary human airway cells with ER stress resulting from cystic fibrosis

Effect of TNF-α in c-Myc deficient HUVECs Pro-inflammatory Response.

<p>Control and Knockdown HUVECs were treated with TNF-α for three hours and analyzed for expression of pro-inflammatory genes by RT-PCR. Results are expressed as fold-change relative to NS-Control. Data were normalized to at least two endogenous control genes. *p<0.05, **p<0.005. ***p<0.0005, (n = 3).</p

Protein Expression Analysis of Pro-Inflammatory Markers.

<p><b>A.</b> Representative western blot image showing time-dependent changes in the expression of Tnfsf15, Vcam1, and Stat1 after c-Myc knockdown. Expression of Stat1 was analyzed 6 days after knockdown. Actin was used as loading control (n = 4). <b>B.</b> Expression analysis of Angptl4, Tgfb2 and Cxcl12 in cell lysates and concentrated supernatants (Spnt) by ELISA 6 days after knockdown (n = 4). NS, control; KD, knockdown. *p<0.05, **p<0.005.</p

Effect of c-Myc Knockdown in Endothelial Cell Proliferation and Morphogenesis.

<p><b>A.</b> Time-course analysis of DNA synthesis in control and knockdown cells from 2–16 hours (n = 3–7). <b>B.</b> Representative images of stimulated control and knockdown cells collected 5 hours after incubation in Matrigel. <b>C.</b> Quantitative analysis of tube formation (n = 3–6). <b>D.</b> Quantification of control and knockdown cell adhesion to extracellular-matrix proteins (n = 5). *p<0.05, **p<0.005. NS, control; KD, knockdown; Col, collagen; FN, fibronectin; LN, laminin; TN, tenascin; VN, vitronectin. Magnification = 10X.</p

Gene Expression Analysis of Inflammatory Mediators and Transcription Factors after c-Myc knockdown.

<p><b>A.</b> Time-dependent changes in the expression of pro-inflammatory genes Angptl4, Cxcl12, Mdk, Tgfb2, Tnfsf15 and Vcam1 in endothelial cells three (black bars) and six (gray bars) days after c-Myc knockdown. <b>B.</b> Transcription factor gene expression profiling 6 days after c-Myc knockdown. Results are expressed as fold-change relative to NS-Control. Gene expression data were normalized to at least two endogenous control genes. <b>C.</b> Network pathway analysis of inflammatory mediators and transcription factors induced by c-Myc knockdown. (n = 4–5) (*p<0.05, **p<0.005).</p