Loss of Smooth Muscle Tenascin-X Inhibits Vascular Remodeling Through Increased TGF-ß Signaling.

BACKGROUND: Vascular smooth muscle cells (VSMCs) are highly plastic. Vessel injury induces a phenotypic transformation from differentiated to dedifferentiated VSMCs, which involves reduced expression of contractile proteins and increased production of extracellular matrix and inflammatory cytokines. This transition plays an important role in several cardiovascular diseases such as atherosclerosis, hypertension, and aortic aneurysm. TGF-ß (transforming growth factor-ß) is critical for VSMC differentiation and to counterbalance the effect of dedifferentiating factors. However, the mechanisms controlling TGF-ß activity and VSMC phenotypic regulation under in vivo conditions are poorly understood. The extracellular matrix protein TN-X (tenascin-X) has recently been shown to bind TGF-ß and to prevent it from activating its receptor. METHODS: We studied the role of TN-X in VSMCs in various murine disease models using tamoxifen-inducible SMC-specific knockout and adeno-associated virus-mediated knockdown. RESULTS: In hypertensive and high-fat diet-fed mice, after carotid artery ligation as well as in human aneurysmal aortae, expression of Tnxb, the gene encoding TN-X, was increased in VSMCs. Mice with smooth muscle cell-specific loss of TN-X (SMC-Tnxb-KO) showed increased TGF-ß signaling in VSMCs, as well as upregulated expression of VSMC differentiation marker genes during vascular remodeling compared with controls. SMC-specific TN-X deficiency decreased neointima formation after carotid artery ligation and reduced vessel wall thickening during Ang II (angiotensin II)-induced hypertension. SMC-Tnxb-KO mice lacking ApoE showed reduced atherosclerosis and Ang II-induced aneurysm formation under high-fat diet. Adeno-associated virus-mediated SMC-specific expression of short hairpin RNA against Tnxb showed similar beneficial effects. Treatment with an anti-TGF-ß antibody or additional SMC-specific loss of the TGF-ß receptor reverted the effects of SMC-specific TN-X deficiency. CONCLUSIONS: In summary, TN-X critically regulates VSMC plasticity during vascular injury by inhibiting TGF-ß signaling. Our data indicate that inhibition of vascular smooth muscle TN-X may represent a strategy to prevent and treat pathological vascular remodeling.

Large chemokine binding spectrum of human and mouse atypical chemokine receptor GPR182 (ACKR5).

Atypical chemokine receptors (ACKRs) play pivotal roles in immune regulation by binding chemokines and regulating their spatial distribution without inducing G-protein activation. Recently, GPR182, provisionally named ACKR5, was identified as a novel ACKR expressed in microvascular and lymphatic endothelial cells, with functions in hematopoietic stem cell homeostasis. Here, we comprehensively investigated the chemokine binding profile of human and mouse GPR182. Competitive binding assays using flow cytometry revealed that besides CXCL10, CXCL12 and CXCL13, also human and mouse CXCL11, CXCL14 and CCL25, as well as human CCL1, CCL11, CCL19, CCL26, XCL1 and mouse CCL22, CCL24, CCL27 and CCL28 bind with an affinity of less than 100 nM to GPR182. In line with the binding affinity observed in vitro, elevated serum levels of CCL22, CCL24, CCL25, and CCL27 were observed in GPR182-deficient mice, underscoring the role of GPR182 in chemokine scavenging. These data show a broader chemokine binding repertoire of GPR182 than previously reported and they will be important for future work exploring the physiological and pathophysiological roles of GPR182, which we propose to be renamed atypical chemokine receptor 5 (ACKR5).

Suppression of CCL2 angiocrine function by adrenomedullin promotes tumor growth.

Within the tumor microenvironment, tumor cells and endothelial cells regulate each other. While tumor cells induce angiogenic responses in endothelial cells, endothelial cells release angiocrine factors, which act on tumor cells and other stromal cells. We report that tumor cell-derived adrenomedullin has a pro-angiogenic as well as a direct tumor-promoting effect, and that endothelium-derived CC chemokine ligand 2 (CCL2) suppresses adrenomedullin-induced tumor cell proliferation. Loss of the endothelial adrenomedullin receptor CALCRL or of the G-protein Gs reduced endothelial proliferation. Surprisingly, tumor cell proliferation was also reduced after endothelial deletion of CALCRL or Gs. We identified CCL2 as a critical angiocrine factor whose formation is inhibited by adrenomedullin. Furthermore, CCL2 inhibited adrenomedullin formation in tumor cells through its receptor CCR2. Consistently, loss of endothelial CCL2 or tumor cell CCR2 normalized the reduced tumor growth seen in mice lacking endothelial CALCRL or Gs. Our findings show tumor-promoting roles of adrenomedullin and identify CCL2 as an angiocrine factor controlling adrenomedullin formation by tumor cells.

GPR182 is an endothelium-specific atypical chemokine receptor that maintains hematopoietic stem cell homeostasis.

G protein-coupled receptor 182 (GPR182) has been shown to be expressed in endothelial cells; however, its ligand and physiological role has remained elusive. We found GPR182 to be expressed in microvascular and lymphatic endothelial cells of most organs and to bind with nanomolar affinity the chemokines CXCL10, CXCL12, and CXCL13. In contrast to conventional chemokine receptors, binding of chemokines to GPR182 did not induce typical downstream signaling processes, including Gq- and Gi-mediated signaling or ß-arrestin recruitment. GPR182 showed relatively high constitutive activity in regard to ß-arrestin recruitment and rapidly internalized in a ligand-independent manner. In constitutive GPR182-deficient mice, as well as after induced endothelium-specific loss of GPR182, we found significant increases in the plasma levels of CXCL10, CXCL12, and CXCL13. Global and induced endothelium-specific GPR182-deficient mice showed a significant decrease in hematopoietic stem cells in the bone marrow as well as increased colony-forming units of hematopoietic progenitors in the blood and the spleen. Our data show that GPR182 is a new atypical chemokine receptor for CXCL10, CXCL12, and CXCL13, which is involved in the regulation of hematopoietic stem cell homeostasis.

Disturbed flow-induced Gs-mediated signaling protects against endothelial inflammation and atherosclerosis.

Atherosclerosis develops preferentially in areas of the arterial system, in which blood flow is disturbed. Exposure of endothelial cells to disturbed flow has been shown to induce inflammatory signaling, including NF-κB activation, which leads to the expression of leukocyte adhesion molecules and chemokines. Here, we show that disturbed flow promotes the release of adrenomedullin from endothelial cells, which in turn activates its Gs-coupled receptor calcitonin receptor-like receptor (CALCRL). This induces antiinflammatory signaling through cAMP and PKA, and it results in reduced endothelial inflammation in vitro and in vivo. Suppression of endothelial expression of Gαs, the α subunit of the G-protein Gs; CALCRL; or adrenomedullin leads to increased disturbed flow-induced inflammatory signaling in vitro and in vivo. Furthermore, mice with induced endothelial-specific deficiency of Gαs, CALCRL, or adrenomedullin show increased atherosclerotic lesions. Our data identify an antiinflammatory signaling pathway in endothelial cells stimulated by disturbed flow and suggest activation of the endothelial adrenomedullin/CALCRL/Gs system as a promising approach to inhibit progression of atherosclerosis.

Identification and validation of mRNA 3'untranslated regions of DNMT3B and TET3 as novel competing endogenous RNAs of the tumor suppressor PTEN.

PTEN inactivation is a frequent event in oncogenesis. Multiple regulatory mechanisms such as promoter hypermethylation, antisense regulation, histone modifications, targeting by microRNAs (miRNAs/miRs) and regulation by transcription factors have all been shown to affect the tumor suppressor functions of PTEN. More recently, the functional involvement of competing endogenous RNAs (ceRNAs) in miRNA­dependent and coding­independent regulation of genes shed light on the highly nuanced control of PTEN expression. The present study has identified and validated DNA methyltransferase 3ß (DNMT3B) and TET methylcytosine dioxygenase 3 (TET3) as novel ceRNAs of PTEN, with which they share multiple miRNAs, in HCT116 colorectal cancer cells. miR­4465 was identified and characterized as a miRNA that directly targets and regulates all 3 transcripts via their 3'untranslated regions (3'UTRs) through a combination of luciferase reporter assays, abrogation of miRNA response elements (MREs) via site­directed mutagenesis, target protection of MREs with locked nucleic acids, RT­qPCR assays and western blot analysis. Competitive miRNA sequestration was demonstrated upon reciprocal 3'UTR overexpression and siRNA­mediated knockdown of their respective transcripts. Overexpression of DNMT3B or TET3 3'UTR promoted apoptosis and decreased migratory capacity, potentially because of shared miRNA sequestration and subsequent activation of PTEN expression. Knockdown of TET3 and DNMT3B decoupled their protein­coding from miRNA­dependent, coding­independent functions. Furthermore, the findings suggested that the phenotypic outcome of ceRNAs is dictated largely by the number of shared miRNAs, and predictably, by the existence of other ceRNA networks in which they participate. Taken together, the findings of the present study identified DNMT3B and TET3 as novel ceRNAs of PTEN that may impact its dose­sensitive tumor suppressive function.