A Type 2 Deiodinase-Dependent Increase in Vegfa Mediates Myoblast-Endothelial Cell Crosstalk During Skeletal Muscle Regeneration

Abstract

Background: The type 2 deiodinase (DIO2) converts thyroxine to 3,3¢,5-triiodothyronine (T3), modulating intracellular T3. An increase in DIO2 within muscle stem cells during skeletal muscle regeneration leads to T3dependent potentiation of differentiation. The muscle stem cell niche comprises numerous cell types, which coordinate the regeneration process. For example, muscle stem cells provide secretory signals stimulating endothelial cell-mediated vascular repair, and, in turn, endothelial cells promote muscle stem differentiation. We hypothesized that Dio2 loss in muscle stem cells directly impairs muscle stem cell-endothelial cell communication, leading to downstream disruption of endothelial cell function. Methods: We assessed the production of proangiogenic factors in differentiated C2C12 cells and in a C2C12 cell line without Dio2 (D2KO C2C12) by real-time quantitative-polymerase chain reaction and enzyme-linked immunosorbent assay. Conditioned medium (CM) was collected daily in parallel to evaluate its effects on human umbilical vein endothelial cell (HUVEC) proliferation, migration and chemotaxis, and vascular network formation. The effects of T3-treatment on vascular endothelial growth factor (Vegfa) mRNA expression in C2C12 cells and mouse muscle were assessed. Chromatin immunoprecipitation (ChIP) identified thyroid hormone receptor (TR) binding to the Vegfa gene. Using mice with a targeted disruption of Dio2 (D2KO mice), we determined endothelial cell number by immunohistochemistry/flow cytometry and evaluated related gene expression in both uninjured and injured skeletal muscle. Results: In differentiated D2KO C2C12 cells, Vegfa expression was 46% of wildtype (WT) C2C12 cells, while secreted VEGF was 45%. D2KO C2C12 CM exhibited significantly less proangiogenic effects on HUVECs. In vitro and in vivo T3 treatment of C2C12 cells and WT mice, and ChIP using antibodies against TRa, indicated that Vegfa is a direct genomic T3 target. In uninjured D2KO soleus muscle, Vegfa expression was decreased by 28% compared with WT mice, while endothelial cell numbers were decreased by 48%. Seven days after skeletal muscle injury, D2KO mice had 36% fewer endothelial cells, coinciding with an 83% decrease in Vegfa expression in fluorescence-activated cell sorting purified muscle stem cells. Conclusion: Dio2 loss in the muscle stem cell impairs muscle stem cell-endothelial cell crosstalk via changes in the T3-responsive gene Vegfa, leading to downstream impairment of endothelial cell function both in vitro and in vivo.

Selective Inhibition of Genomic and Non-Genomic Effects of Thyroid Hormone Regulates Muscle Cell Differentiation and Metabolic Behavior

“…When interfered with for D2, muscle cells showed an enhanced proliferation rate and reduced cell differentiation ability. These results confirm the previous findings that the D2-mediated TH activation fosters muscle cell differentiation while attenuating myotube formation [15,[25][26][27]. Similarly, in different tissue compartments, TH acts as a pro-differentiation agent [28,29].…”

Thyroid Hormone Enhances Angiogenesis and the Warburg Effect in Squamous Cell Carcinomas

“…Recently, VEGF-A was found to be a positive TH target gene in C2C12 muscle cells, in which TH promotes VEGF-A secretion and VEGF-A-mediated stimulation of muscular endothelial cells [ 34 ]. Given the tumor-promoting effects of TH in SCC tumor progression [ 16 ], we asked if, similar to skeletal muscle, TH induces VEGF-A in SCC cells.…”

“…Contextually, the reverse effects are promoted by TH, which stimulates VEGF-A and VEGFR2 expression by endothelial cells ( Figure S2D,E ). These effects exerted by TH on VEGF-A expression resemble those observed in muscle cells in which TH-mediated VEGF-A production regulates the muscle stem cell–endothelial cell communication and leads to downstream induction of EC function [ 34 ]. Furthermore, these data are consistent with the previous finding that TH promotes angiogenesis in a chick chorioallantoic membrane model and that D2 is expressed in the tumor stroma of colon rectal cancer cells [ 54 , 55 , 56 ].…”

Cross-Talk between the Cytokine IL-37 and Thyroid Hormones in Modulating Chronic Inflammation Associated with Target Organ Damage in Age-Related Metabolic and Vascular Conditions

“…Broadly, a fibrotic matrix forms progressively, whereby senescent, secretory, and proinflammatory cells accumulate in many tissues, often in conjunction with profound immune alterations [ 61 ]. The age-associated inflammation increases vasopermeability and extravasation of plasma constituents, changing the ability of THs to cross the endothelial barrier and access target tissues [ 41 , 62 , 63 ]. Thus, the exact fraction of fT4/fT3 that goes into the extracellular matrix and reaches cellular targets is likely to depend on the environment.…”