Subramaniam, Malayannan

Background: Endothelial progenitor cells (EPCs) may originate from the bone marrow and are capable of contributing to cardiovascular regeneration. However, the identities of these bone marrow cells and the signaling pathways that regulate their differentiation into functional EPCs remain poorly understood. Hematopoiesis consists of a cascade by which hematopoietic stem cells (HSCs) give rise to either a common myeloid progenitor (CMPs) or common lymphoid progenitor (CLPs). The CMP cells can give rise to either granulocyte-monocyte progenitors (GMPs) or megakaryocyte-erythrocyte progenitors (MEPs). Methods and Results: Herein, we identify that CMPs and GMPs preferentially differentiate into CD34+KDR+ EPCs as measured by FACS, uptake of DiIacLDL and U.lex Lectin, matrigel studies, and endothelial markers. Co-culture studies in matrigel demonstrate increased network formation and incorporation when HUVECs were co-incubated with CMPs and GMPs only. Treatment of CMPs or GMPs with IFN-g blocked EPC differentiation, whereas treatment with TGF-b1 potently induced EPC differentiation. Conversely, EPC formation was markedly impaired using blocking antibodies or TGF-b1 deficient CMPs or GMPs, whereas IFN-g deficient mice have increased EPC differentiation. Functionally, EPCs derived from CMPs and GMPs more potently adhered to fibronectin-coated plates, migrated faster in transwell Boyden chambers, and released VEGF nearly ~2 to 2.5-fold higher than EPCs from MEPs and HSCs. Finally, we identified that a TGF-b1-responsive Kruppel-like transcription factor, KLF10, as robustly expressed ~60-fold in EPCs derived from CMPs and GMPs, compared to hematopoietic progenitors lacking EPC markers. Consistently, KLF10−/− mice have marked defects in EPC differentiation (−23.6% vs. WT, P<0.002), TGF-b1 responsiveness (completedly abolished), and impaired blood flow recovery in response to hindlimb ischemia (−23.8% vs. WT, P<0.008), an effect associated with reduced levels of circulating EPCs (−28.2% vs. WT, P<0.05). Conclusion: Among bone marrow progenitors, a hierarchy exists for EPC differentiation. Targeting KLF10 may alter EPC differentiation from bone marrow-derived CMPs and GMPs and influence cardiovascular repair.

The transcription factor Krüppel-like factor 10 (Klf10), also known as Tieg1 for TGFβ (Inducible Early Gene-1) is known to control numerous genes in many cell types that are involved in various key biological processes (differentiation, proliferation, apoptosis, inflammation), including cell metabolism and human disease. In skeletal muscle, particularly in the soleus, deletion of the Klf10 gene (Klf10 KO) resulted in ultrastructure fiber disorganization and mitochondrial metabolism deficiencies, characterized by muscular hypertrophy. To determine the metabolic profile related to loss of Klf10 expression, we analyzed blood and soleus tissue using UHPLC-Mass Spectrometry. Metabolomics analyses on both serum and soleus revealed profound differences between wild-type (WT) and KO animals. Klf10 deficient mice exhibited alterations in metabolites associated with energetic metabolism. Additionally, chemical classes of aromatic and amino-acid compounds were disrupted, together with Krebs cycle intermediates, lipids and phospholipids. From variable importance in projection (VIP) analyses, the Warburg effect, citric acid cycle, gluconeogenesis and transfer of acetyl groups into mitochondria appeared to be possible pathways involved in the metabolic alterations observed in Klf10 KO mice. These studies have revealed essential roles for Klf10 in regulating multiple metabolic pathways whose alterations may underlie the observed skeletal muscle defects as well as other diseases.

AbstractIntroduction/AimsKlf10 is a member of the Krüppel‐like family of transcription factors, which is implicated in mediating muscle structure (fiber size, organization of the sarcomere), muscle metabolic activity (respiratory chain), and passive force. The aim of this study was to further characterize the roles of Klf10 in the contractile properties of skeletal muscle fibers.MethodsFifty‐two single fibers were extracted from female wild‐type (WT) and Klf10 knockout (KO) oxidative (soleus) and glycolytic (extensor digitorum longus [EDL]) skinned muscles. Each fiber was immersed successively in relaxing (R), washing (W), and activating (A) solutions. Calcium was included in the activating solution to induce a maximum contraction of the fiber. The maximum force (Fmax) was measured and normalized to the cross‐sectional area to obtain the maximum stress (Stressmax). After a steady state in contraction was reached, a quick stretch‐release was performed; the force at the maximum stretch (Fstretch) was measured and the stiffness was assessed.ResultsDeletion of the Klf10 gene induced changes in the contractile parameters (Fmax, Stressmax, Stiffness), which were lower and higher for soleus and EDL fibers compared with littermates, respectively. These measurements also revealed changes in the proportion and resistance of attached cross‐bridges.DiscussionKlf10 plays a major role in the homeostasis of the contractile behavior of skeletal muscle fibers in a muscle fiber type–specific manner. These findings further implicate important roles for Klf10 in skeletal muscle function and shed new light on understanding the molecular processes regulating the contractility of skeletal muscle fibers.

The mammalian circadian timing system and metabolism are highly interconnected, and disruption of this coupling is associated with negative health outcomes. Krüppel-like factors (KLFs) are transcription factors that govern metabolic homeostasis in various organs. Many KLFs show a circadian expression in the liver. Here, we show that the loss of the clock-controlled KLF10 in hepatocytes results in extensive reprogramming of the mouse liver circadian transcriptome, which in turn alters the temporal coordination of pathways associated with energy metabolism. We also show that glucose and fructose induce Klf10, which helps mitigate glucose intolerance and hepatic steatosis in mice challenged with a sugar beverage. Functional genomics further reveal that KLF10 target genes are primarily involved in central carbon metabolism. Together, these findings show that in the liver KLF10 integrates circadian timing and sugar metabolism-related signaling, and serves as a transcriptional brake that protects against the deleterious effects of increased sugar consumption.

We have previously demonstrated that β-catenin plays important roles in valve calcification with a specific osteogenic phenotype defined by increased bone mineral content and overall valve thickening. Recent studies indicate that KLF10 may be involved in mediating the Wnt signaling pathway in bone, which is known to play critical roles in osteoblast differentiation and mineralization. Therefore, we sought to test the role of KLF10 in mediating Wnt signaling, as well as differentiation and mineralization, in valve interstitial cells (VICs) isolated from porcine valves. Exposure of VICs to differentiation media led to increased expression of Runx2, Sox9 and osteocalcin. Differentiated cells also stained positive with Von Kossa while undifferentiated cells stained negative confirming the induction of an osteogenic phenotype. As expected, over-expression of both Lef1 and β-catenin led to activation of the top-flash reporter when transfected into VICs. Interestingly, over-expression of KLF10 also significantly up-regulated the top-flash reporter alone and further enhanced the activity of both Lef1 and β-catenin when co-transfected. We further confirmed the role of TIEG in an atherosclerotic mouse model ApoE/LRP5 double KO and found a two-fold increase in KLF10, Lrp6, and Runx2 expression in the cholesterol treated aortic valves as compared to controls. These data suggested that KLF10, Lef1 and β-catenin interact with each other to form a transcriptionally active protein complex leading to enhanced Wnt signaling in VICs. This possibility was further confirmed by the observation that KLF10 and β-catenin co-localize with one another in the nucleus of VICs following stimulation with LiCl and/or TGF-β. Taken together, these data implicate an important role for KLF10 in mediating Wnt signaling and Lef1 transcriptional activity in VICs, and implicate a potential role for canonical Wnt signaling in the observed osteogenic bone phenotype of cardiac aortic valves.

Background— Calcific aortic valve disease is the most common indication for surgical valve replacement in the United States. The cellular mechanisms of valve calcification are not well understood. We have previously shown that cellular proliferation and osteoblastogenesis are important in the development of valvular heart disease. Lrp5, a known low-density receptor-related protein, plays an essential role in cellular proliferation and osteoblastogenesis via the β-catenin signaling pathway. We hypothesize that Lrp5 also plays a role in aortic valve (AV) calcification in experimental hypercholesterolemia. Methods and Results— We examined the effects of cholesterol and atorvastatin in Watanabe rabbits (n=54). Group I (n=18) received a normal diet, group II (n=18) a 0.25% cholesterol diet, and group III (n=18) a 0.25% (w/w) cholesterol diet with atorvastatin for the development of calcification. The AVs were examined for cellular proliferation, Lrp5/β-catenin, and bone matrix markers. Bone formation was assessed by micro-computed tomography, calcein injection, and osteopontin expression. Low-density lipoprotein with and without atorvastatin was also tested in AV myofibroblasts for cellular proliferation and regulation of the Lrp5/β-catenin pathway. Our results demonstrate that the cholesterol diet induced complex bone formations in the calcified AVs with an increase in the Lrp5 receptors, osteopontin, and p42/44 expression. Atorvastatin reduced bone formation, cellular proliferation, and Lrp5/β-catenin protein levels in the AVs. In vitro analysis confirmed the Lrp5/β-catenin expression in myofibroblast cell proliferation. Conclusion— Hypercholesterolemic AV calcification is attenuated by atorvastatin and is mediated in part by the Lrp5/β-catenin pathway. This developmental pathway may be important in the signaling pathway of this disease.

Abstract Despite the availability of drugs that target ERα-positive breast cancer, resistance commonly occurs, resulting in relapse, metastasis, and death. Tamoxifen remains the most commonly-prescribed endocrine therapy worldwide, and “tamoxifen resistance” has been extensively studied. However, little consideration has been given to the role of endoxifen, the most abundant active tamoxifen metabolite detected in patients, in driving resistance mechanisms. Endoxifen functions differently from the parent drug and other primary metabolites, including 4-hydroxy-tamoxifen (4HT). Many studies have shown that patients who extensively metabolize tamoxifen into endoxifen have superior outcomes relative to patients who do not, supporting a primary role for endoxifen in driving tamoxifen responses. Therefore, “tamoxifen resistance” may be better modeled by “endoxifen resistance” for some patients. Here, we report the development of novel endoxifen-resistant breast cancer cell lines and have extensively compared these models to 4HT and fulvestrant (ICI)-resistant models. Endoxifen-resistant cells were phenotypically and molecularly distinct from 4HT-resistant cells and more closely resembled ICI-resistant cells overall. Specifically, endoxifen resistance was associated with ERα and PR loss, estrogen insensitivity, unique gene signatures, and striking resistance to most FDA-approved second- and third-line therapies. Given these findings, and the importance of endoxifen in the efficacy of tamoxifen therapy, our data indicate that endoxifen-resistant models may be more clinically relevant than existing models and suggest that a better understanding of endoxifen resistance could substantially improve patient care. Implications: Here we report on the development and characterization of the first endoxifen-resistant models and demonstrate that endoxifen resistance may better model tamoxifen resistance in a subset of patients.