MicroRNA-409-3p/BTG2 signaling axis improves impaired angiogenesis and wound healing in obese mice.

Wound healing is facilitated by neoangiogenesis, a complex process that is essential to tissue repair in response to injury. MicroRNAs are small, noncoding RNAs that can regulate the wound healing process including stimulation of impaired angiogenesis that is associated with type-2 diabetes (T2D). Expression of miR-409-3p was significantly increased in the nonhealing skin wounds of patients with T2D compared to the non-wounded normal skin, and in the skin of a murine model with T2D. In response to high glucose, neutralization of miR-409-3p markedly improved EC growth and migration in human umbilical vein endothelial cells (HUVECs), promoted wound closure and angiogenesis as measured by increased CD31 in human skin organoids, while overexpression attenuated EC angiogenic responses. Bulk mRNA-Seq transcriptomic profiling revealed BTG2 as a target of miR-409-3p, where overexpression of miR-409-3p significantly decreased BTG2 mRNA and protein expression. A 3' untranslated region (3'-UTR) luciferase assay of BTG2 revealed decreased luciferase activity with overexpression of miR-409-3p, while inhibition had opposite effects. Mechanistically, in response to high glucose, miR-409-3p deficiency in ECs resulted in increased mTOR phosphorylation, meanwhile BTG-anti-proliferation factor 2 (BTG2) silencing significantly decreased mTOR phosphorylation. Endothelial-specific and tamoxifen-inducible miR-409-3p knockout mice (MiR-409IndECKO ) with hyperglycemia that underwent dorsal skin wounding showed significant improvement of wound closure, increased blood flow, granulation tissue thickness (GTT), and CD31 that correlated with increased BTG2 expression. Taken together, our results show that miR-409-3p is a critical mediator of impaired angiogenesis in diabetic skin wound healing.

MiR-409-3p targets a MAP4K3-ZEB1-PLGF signaling axis and controls brown adipose tissue angiogenesis and insulin resistance.

Endothelial cells (ECs) within the microvasculature of brown adipose tissue (BAT) are important in regulating the plasticity of adipocytes in response to increased metabolic demand by modulating the angiogenic response. However, the mechanism of EC-adipocyte crosstalk during this process is not completely understood. We used RNA sequencing to profile microRNAs derived from BAT ECs of obese mice and identified an anti-angiogenic microRNA, miR-409-3p. MiR-409-3p overexpression inhibited EC angiogenic properties; whereas, its inhibition had the opposite effects. Mechanistic studies revealed that miR-409-3p targets ZEB1 and MAP4K3. Knockdown of ZEB1/MAP4K3 phenocopied the angiogenic effects of miR-409-3p. Adipocytes co-cultured with conditioned media from ECs deficient in miR-409-3p showed increased expression of BAT markers, UCP1 and CIDEA. We identified a pro-angiogenic growth factor, placental growth factor (PLGF), released from ECs in response to miR-409-3p inhibition. Deficiency of ZEB1 or MAP4K3 blocked the release of PLGF from ECs and PLGF stimulation of 3T3-L1 adipocytes increased UCP1 expression in a miR-409-3p dependent manner. MiR-409-3p neutralization improved BAT angiogenesis, glucose and insulin tolerance, and energy expenditure in mice with diet-induced obesity. These findings establish miR-409-3p as a critical regulator of EC-BAT crosstalk by modulating a ZEB1-MAP4K3-PLGF signaling axis, providing new insights for therapeutic intervention in obesity.

MiR-4674 regulates angiogenesis in tissue injury by targeting p38K signaling in endothelial cells.

Neoangiogenesis is critical for tissue repair in response to injury such as myocardial ischemia or dermal wound healing. MicroRNAs are small noncoding RNAs and important regulators of angiogenesis under physiological and pathological disease states. Therefore, identification of microRNAs that may restore impaired angiogenesis in response to tissue injury may provide new targets for therapy. Using a microRNA microarray profiling approach, we identified a human-specific microRNA, miR-4674, that was significantly decreased in patients after myocardial tissue injury and had an endothelial cell (EC)-enriched expression pattern. Functionally, overexpression of miR-4674 markedly attenuated EC proliferation, migration, network tube formation, and spheroid sprouting, whereas blockade of miR-4674 had the opposite effects. Transcriptomic profiling, gene set enrichment analyses, bioinformatics, 3'-untranslated region (3'-UTR) reporter and microribonucleoprotein immunoprecipitation (miRNP-IP) assays, and small interfering RNA dependency studies revealed that miR-4674 regulates VEGF stimulated-p38 mitogen-activated protein kinase (MAPK) signaling and targets interleukin 1 receptor-associated kinase 1 (Irak1) and BICD cargo adaptor 2 (Bicd2) in ECs. Furthermore, Irak1 and Bicd2 were necessary for miR-4674-driven EC proliferation and migration. Finally, neutralization of miR-4674 increased angiogenesis, Irak1 and Bicd2 expression, and p38 phosphorylation in human skin organoids as a model of tissue injury. Collectively, targeting miR-4674 may provide a novel therapeutic target for tissue repair in pathological disease states associated with impaired angiogenesis.

MicroRNA-135a-3p regulates angiogenesis and tissue repair by targeting p38 signaling in endothelial cells.

Angiogenesis is a critical process in repair of tissue injury that is regulated by a delicate balance between pro- and antiangiogenic factors. In disease states associated with impaired angiogenesis, we identified that miR-135a-3p is rapidly induced and serves as an antiangiogenic microRNA (miRNA) by targeting endothelial cell (EC) p38 signaling in vitro and in vivo. MiR-135a-3p overexpression significantly inhibited EC proliferation, migration, and network tube formation in matrigel, whereas miR-135-3p neutralization had the opposite effects. Mechanistic studies using transcriptomic profiling, bioinformatics, 3'-UTR reporter and miRNA ribonucleoprotein complex -immunoprecipitation assays, and small interfering RNA dependency studies revealed that miR-135a-3p inhibits the p38 signaling pathway in ECs by targeting huntingtin-interacting protein 1 (HIP1). Local delivery of miR-135a-3p inhibitors to wounds of diabetic db/db mice markedly increased angiogenesis, granulation tissue thickness, and wound closure rates, whereas local delivery of miR-135a-3p mimics impaired these effects. Finally, through gain- and loss-of-function studies in human skin organoids as a model of tissue injury, we demonstrated that miR-135a-3p potently modulated p38 signaling and angiogenesis in response to VEGF stimulation by targeting HIP1. These findings establish miR-135a-3p as a pivotal regulator of pathophysiological angiogenesis and tissue repair by targeting a VEGF-HIP1-p38K signaling axis, providing new targets for angiogenic therapy to promote tissue repair.-Icli, B., Wu, W., Ozdemir, D., Li, H., Haemmig, S., Liu, X., Giatsidis, G., Cheng, H. S., Avci, S. N., Kurt, M., Lee, N., Guimaraes, R. B., Manica, A., Marchini, J. F., Rynning, S. E., Risnes, I., Hollan, I., Croce, K., Orgill, D. P., Feinberg, M. W. MicroRNA-135a-3p regulates angiogenesis and tissue repair by targeting p38 signaling in endothelial cells.

MicroRNA-615-5p Regulates Angiogenesis and Tissue Repair by Targeting AKT/eNOS (Protein Kinase B/Endothelial Nitric Oxide Synthase) Signaling in Endothelial Cells.

Objective- In response to tissue injury, the appropriate progression of events in angiogenesis is controlled by a careful balance between pro and antiangiogenic factors. We aimed to identify and characterize microRNAs that regulate angiogenesis in response to tissue injury. Approach and Results- We show that in response to tissue injury, microRNA-615-5p (miR-615-5p) is rapidly induced and serves as an antiangiogenic microRNA by targeting endothelial cell VEGF (vascular endothelial growth factor)-AKT (protein kinase B)/eNOS (endothelial nitric oxide synthase) signaling in vitro and in vivo. MiR-615-5p expression is increased in wounds of diabetic db/db mice, in plasma of human subjects with acute coronary syndromes, and in plasma and skin of human subjects with diabetes mellitus. Ectopic expression of miR-615-5p markedly inhibited endothelial cell proliferation, migration, network tube formation in Matrigel, and the release of nitric oxide, whereas miR-615-5p neutralization had the opposite effects. Mechanistic studies using transcriptomic profiling, bioinformatics, 3' untranslated region reporter and microribonucleoprotein immunoprecipitation assays, and small interfering RNA dependency studies demonstrate that miR-615-5p inhibits the VEGF-AKT/eNOS signaling pathway in endothelial cells by targeting IGF2 (insulin-like growth factor 2) and RASSF2 (Ras-associating domain family member 2). Local delivery of miR-615-5p inhibitors, markedly increased angiogenesis, granulation tissue thickness, and wound closure rates in db/db mice, whereas miR-615-5p mimics impaired these effects. Systemic miR-615-5p neutralization improved skeletal muscle perfusion and angiogenesis after hindlimb ischemia in db/db mice. Finally, modulation of miR-615-5p expression dynamically regulated VEGF-induced AKT signaling and angiogenesis in human skin organoids as a model of tissue injury. Conclusions- These findings establish miR-615-5p as an inhibitor of VEGF-AKT/eNOS-mediated endothelial cell angiogenic responses and that manipulating miR-615-5p expression could provide a new target for angiogenic therapy in response to tissue injury. Visual Overview- An online visual overview is available for this article.

MicroRNA-181b Improves Glucose Homeostasis and Insulin Sensitivity by Regulating Endothelial Function in White Adipose Tissue.

RATIONALE: The pathogenesis of insulin resistance involves dysregulated gene expression and function in multiple cell types, including endothelial cells (ECs). Post-transcriptional mechanisms such as microRNA-mediated regulation of gene expression could affect insulin action by modulating EC function. OBJECTIVE: To determine whether microRNA-181b (miR-181b) affects the pathogenesis of insulin resistance by regulating EC function in white adipose tissue during obesity. METHODS AND RESULTS: MiR-181b expression was reduced in adipose tissue ECs of obese mice, and rescue of miR-181b expression improved glucose homeostasis and insulin sensitivity. Systemic intravenous delivery of miR-181b robustly accumulated in adipose tissue ECs, enhanced insulin-mediated Akt phosphorylation at Ser473, and reduced endothelial dysfunction, an effect that shifted macrophage polarization toward an M2 anti-inflammatory phenotype in epididymal white adipose tissue. These effects were associated with increased endothelial nitric oxide synthase and FoxO1 phosphorylation as well as nitric oxide activity in epididymal white adipose tissue. In contrast, miR-181b did not affect insulin-stimulated Akt phosphorylation in liver and skeletal muscle. Bioinformatics and gene profiling approaches revealed that Pleckstrin homology domain leucine-rich repeat protein phosphatase, a phosphatase that dephosphorylates Akt at Ser473, is a novel target of miR-181b. Knockdown of Pleckstrin homology domain leucine-rich repeat protein phosphatase increased Akt phosphorylation at Ser473 in ECs, and phenocopied miR-181b's effects on glucose homeostasis, insulin sensitivity, and inflammation of epididymal white adipose tissue in vivo. Finally, ECs from diabetic subjects exhibited increased Pleckstrin homology domain leucine-rich repeat protein phosphatase expression. CONCLUSIONS: Our data underscore the importance of adipose tissue EC function in controlling the development of insulin resistance. Delivery of miR-181b or Pleckstrin homology domain leucine-rich repeat protein phosphatase inhibitors may represent a new therapeutic approach to ameliorate insulin resistance by improving adipose tissue endothelial Akt-endothelial nitric oxide synthase-nitric oxide signaling.

MicroRNA-181b inhibits thrombin-mediated endothelial activation and arterial thrombosis by targeting caspase recruitment domain family member 10.

Thrombogenic and inflammatory mediators, such as thrombin, induce NF-κB-mediated endothelial cell (EC) activation and dysfunction, which contribute to pathogenesis of arterial thrombosis. The role of anti-inflammatory microRNA-181b (miR-181b) on thrombosis remains unknown. Our previous study demonstrated that miR-181b inhibits downstream NF-κB signaling in response to TNF-α. Here, we demonstrate that miR-181b uniquely inhibits upstream NF-κB signaling in response to thrombin. Overexpression of miR-181b inhibited thrombin-induced activation of NF-κB signaling, demonstrated by reduction of phospho-IKK-ß, -IκB-α, and p65 nuclear translocation in ECs. MiR-181b also reduced expression of NF-κB target genes VCAM-1, intercellular adhesion molecule-1, E-selectin, and tissue factor. Mechanistically, miR-181b targets caspase recruitment domain family member 10 (Card10), an adaptor protein that participates in activation of the IKK complex in response to signals transduced from protease-activated receptor-1. miR-181b reduced expression of Card10 mRNA and protein, but not protease-activated receptor-1. 3'-Untranslated region reporter assays, argonaute-2 microribonucleoprotein immunoprecipitation studies, and Card10 rescue studies revealed that Card10 is a bona fide direct miR-181b target. Small interfering RNA-mediated knockdown of Card10 expression phenocopied effects of miR-181b on NF-κB signaling and targets. Card10 deficiency did not affect TNF-α-induced activation of NF-κB signaling, which suggested stimulus-specific regulation of NF-κB signaling and endothelial responses by miR-181b in ECs. Finally, in response to photochemical injury-induced arterial thrombosis, systemic delivery of miR-181b reduced thrombus formation by 73% in carotid arteries and prolonged time to occlusion by 1.6-fold, effects recapitulated by Card10 small interfering RNA. These data demonstrate that miR-181b and Card10 are important regulators of thrombin-induced EC activation and arterial thrombosis. These studies highlight the relevance of microRNA-dependent targets in response to ligand-specific signaling in ECs.-Lin, J., He, S., Sun, X., Franck, G., Deng, Y., Yang, D., Haemmig, S., Wara, A. K. M., Icli, B., Li, D., Feinberg, M. W. MicroRNA-181b inhibits thrombin-mediated endothelial activation and arterial thrombosis by targeting caspase recruitment domain family member 10.

Regulation of impaired angiogenesis in diabetic dermal wound healing by microRNA-26a.

Wound healing is a physiological reparative response to injury and a well-orchestrated process that involves hemostasis, cellular migration, proliferation, angiogenesis, extracellular matrix deposition, and wound contraction and re-epithelialization. However, patients with type 2 diabetes mellitus (T2D) are frequently afflicted with impaired wound healing that progresses into chronic wounds or diabetic ulcers, and may lead to complications including limb amputation. Herein, we investigate the potential role of microRNA-26a (miR-26a) in a diabetic model of wound healing. Expression of miR-26a is rapidly induced in response to high glucose in endothelial cells (ECs). Punch skin biopsy wounding of db/db mice revealed increased expression of miR-26a (~3.5-fold) four days post-wounding compared to that of WT mice. Local administration of a miR-26a inhibitor, LNA-anti-miR-26a, induced angiogenesis (up to ~80%), increased granulation tissue thickness (by 2.5-fold) and accelerated wound closure (53% after nine days) compared to scrambled anti-miR controls in db/db mice. These effects were independent of altered M1/M2 macrophage ratios. Mechanistically, inhibition of miR-26a increased its target gene SMAD1 in ECs nine days post-wounding of diabetic mice. In addition, high glucose reduced activity of the SMAD1-3'-UTR. Diabetic dermal wounds treated with LNA-anti-miR-26a had increased expression of ID1, a downstream modulator or SMAD1, and decreased expression of the cell cycle inhibitor p27. These findings establish miR-26a as an important regulator on the progression of skin wounds of diabetic mice by specifically regulating the angiogenic response after injury, and demonstrate that neutralization of miR-26a may serve as a novel approach for therapy.

Systemic delivery of microRNA-181b inhibits nuclear factor-κB activation, vascular inflammation, and atherosclerosis in apolipoprotein E-deficient mice.

RATIONALE: Activated nuclear factor (NF)-κB signaling in the vascular endothelium promotes the initiation and progression of atherosclerosis. Targeting endothelial NF-κB may provide a novel strategy to limit chronic inflammation. OBJECTIVE: To examine the role of microRNA-181b (miR-181b) in endothelial NF-κB signaling and effects on atherosclerosis. METHODS AND RESULTS: MiR-181b expression was reduced in the aortic intima and plasma in apolipoprotein E-deficient mice fed a high-fat diet. Correspondingly, circulating miR-181b in the plasma was markedly reduced in human subjects with coronary artery disease. Systemic delivery of miR-181b resulted in a 2.3-fold overexpression of miR-181b in the aortic intima of apolipoprotein E-deficient mice and suppressed NF-κB signaling revealed by bioluminescence imaging and reduced target gene expression in the aortic arch in apolipoprotein E-deficient/NF-κB-luciferase transgenic mice. MiR-181b significantly inhibited atherosclerotic lesion formation, proinflammatory gene expression and the influx of lesional macrophages and CD4+ T cells in the vessel wall. Mechanistically, miR-181b inhibited the expression of the target gene importin-α3, an effect that reduced NF-κB nuclear translocation specifically in the vascular endothelium of lesions, whereas surprisingly leukocyte NF-κB signaling was unaffected despite a 7-fold overexpression of miR-181b. Our findings uncover that NF-κB nuclear translocation in leukocytes does not involve importin-α3, but rather importin-α5, which miR-181b does not target, highlighting that inhibition of NF-κB signaling in the endothelium is sufficient to mediate miR-181b's protective effects. CONCLUSIONS: Systemic delivery of miR-181b inhibits the activation of NF-κB and atherosclerosis through cell-specific mechanisms in the vascular endothelium. These findings support the rationale that delivery of miR-181b may provide a novel therapeutic approach to treat chronic inflammatory diseases such as atherosclerosis.

MicroRNA-26a regulates pathological and physiological angiogenesis by targeting BMP/SMAD1 signaling.

RATIONALE: The rapid induction and orchestration of new blood vessels are critical for tissue repair in response to injury, such as myocardial infarction, and for physiological angiogenic responses, such as embryonic development and exercise. OBJECTIVE: We aimed to identify and characterize microRNAs (miR) that regulate pathological and physiological angiogenesis. METHODS AND RESULTS: We show that miR-26a regulates pathological and physiological angiogenesis by targeting endothelial cell (EC) bone morphogenic protein/SMAD1 signaling in vitro and in vivo. MiR-26a expression is increased in a model of acute myocardial infarction in mice and in human subjects with acute coronary syndromes. Ectopic expression of miR-26a markedly induced EC cycle arrest and inhibited EC migration, sprouting angiogenesis, and network tube formation in matrigel, whereas blockade of miR-26a had the opposite effects. Mechanistic studies demonstrate that miR-26a inhibits the bone morphogenic protein/SMAD1 signaling pathway in ECs by binding to the SMAD1 3'-untranslated region, an effect that decreased expression of Id1 and increased p21(WAF/CIP) and p27. In zebrafish, miR-26a overexpression inhibited formation of the caudal vein plexus, a bone morphogenic protein-responsive process, an effect rescued by ectopic SMAD1 expression. In mice, miR-26a overexpression inhibited EC SMAD1 expression and exercise-induced angiogenesis. Furthermore, systemic intravenous administration of an miR-26a inhibitor, locked nucleic acid-anti-miR-26a, increased SMAD1 expression and rapidly induced robust angiogenesis within 2 days, an effect associated with reduced myocardial infarct size and improved heart function. CONCLUSIONS: These findings establish miR-26a as a regulator of bone morphogenic protein/SMAD1-mediated EC angiogenic responses, and that manipulating miR-26a expression could provide a new target for rapid angiogenic therapy in ischemic disease states.

Bone marrow-derived Kruppel-like factor 10 controls reendothelialization in response to arterial injury.

OBJECTIVE: The objective of this study was to investigate the role of Kruppel-like factor (KLF) 10, a zinc-finger transcription factor, in bone marrow (BM)-derived cell responses to arterial endothelial injury. Accumulating evidence indicates that BM-derived progenitors are recruited to sites of vascular injury and contribute to endothelial repair. APPROACH AND RESULTS: In response to carotid artery endothelial denudation, KLF10 mRNA expression was markedly increased in both BM and circulating lin(-) progenitor cells. To examine the specific role of KLF10 in arterial reendothelialization, we used 2 models of endothelial denudation (wire- and thermal-induced injury) of the carotid artery in wild-type (WT) and KLF10(-/-) mice. WT mice displayed higher areas of reendothelialization compared with KLF10(-/-) mice after endothelial injury using either method. BM transplant studies revealed that reconstitution of KLF10(-/-) mice with WT BM fully rescued the defect in reendothelialization and increased lin(-)CD34(+)kinase insert domain receptor(+) progenitors in the blood and injured carotid arteries. Conversely, reconstitution of WT mice with KLF10(-/-) BM recapitulated the defects in reendothelialization and peripheral cell progenitors. The media from cultured KLF10(-)/(-) BM progenitors was markedly inefficient in promoting endothelial cell growth and migration compared with the media from WT progenitors, indicative of defective paracrine trophic effects from KLF10(-)/(-) BM progenitors. Finally, BM-derived KLF10(-/-) lin(-) progenitors from reconstituted mice had reduced CXC-chemokine receptor 4 expression and impaired migratory responses. CONCLUSIONS: Collectively, these observations demonstrate a protective role for BM-derived KLF10 in paracrine and homing responses important for arterial endothelial injury and highlight KLF10 as a possible therapeutic target to promote endothelial repair in vascular disease states.

Bone marrow-derived CMPs and GMPs represent highly functional proangiogenic cells: implications for ischemic cardiovascular disease.

Clinical studies using bone marrow-derived proangiogenic cells (PACs) have demonstrated modest improvements of function and/or perfusion of ischemic myocardium or skeletal muscle. Because the identities of these PACs and their functional ability to promote neovascularization remain poorly understood, it is possible that a subset of robust PACs exists but is obscured by the heterogeneous nature of this cell population. Herein, we found that common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) preferentially differentiate into PACs compared with megakaryocyte-erythrocyte progenitors, hematopoietic stem cells, and common lymphoid progenitors. In vivo hindlimb ischemia studies and Matrigel plug assays verified the enhanced neovascularization properties uniquely associated with PACs derived from CMPs and GMPs. Taken together, these observations identify CMPs and GMPs as key bone marrow progenitors for optimal PAC function in vitro and in vivo and provide a foundation for novel therapeutic approaches to modulate angiogenesis.

TGF-ß1 signaling and Krüppel-like factor 10 regulate bone marrow-derived proangiogenic cell differentiation, function, and neovascularization.

Emerging evidence demonstrates that proangiogenic cells (PACs) originate from the BM and are capable of being recruited to sites of ischemic injury where they contribute to neovascularization. We previously determined that among hematopoietic progenitor stem cells, common myeloid progenitors (CMPs) and granulocyte-macrophage progenitor cells (GMPs) differentiate into PACs and possess robust angiogenic activity under ischemic conditions. Herein, we report that a TGF-ß1-responsive Krüppel- like factor, KLF10, is strongly expressed in PACs derived from CMPs and GMPs, ∼ 60-fold higher than in progenitors lacking PAC markers. KLF10(-/-) mice present with marked defects in PAC differentiation, function, TGF-ß responsiveness, and impaired blood flow recovery after hindlimb ischemia, an effect rescued by wild-type PACs, but not KLF10(-/-) PACs. Overexpression studies revealed that KLF10 could rescue PAC formation from TGF-ß1(+/-) CMPs and GMPs. Mechanistically, KLF10 targets the VEGFR2 promoter in PACs which may underlie the observed effects. These findings may be clinically relevant because KLF10 expression was also found to be significantly reduced in PACs from patients with peripheral artery disease. Collectively, these observations identify TGF-ß1 signaling and KLF10 as key regulators of functional PACs derived from CMPs and GMPs and may provide a therapeutic target during cardiovascular ischemic states.

Deficiency of miR-409-3p improves myocardial neovascularization and function through modulation of DNAJB9/p38 MAPK signaling.

Angiogenesis is critical for tissue repair following myocardial infarction (MI), which is exacerbated under insulin resistance or diabetes. MicroRNAs are regulators of angiogenesis. We examined the metabolic regulation of miR-409-3p in post-infarct angiogenesis. miR-409-3p was increased in patients with acute coronary syndrome (ACS) and in a mouse model of acute MI. In endothelial cells (ECs), miR-409-3p was induced by palmitate, while vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) decreased its expression. Overexpression of miR-409-3p decreased EC proliferation and migration in the presence of palmitate, whereas inhibition had the opposite effects. RNA sequencing (RNA-seq) profiling in ECs identified DNAJ homolog subfamily B member 9 (DNAJB9) as a target of miR-409-3p. Overexpression of miR-409-3p decreased DNAJB9 mRNA and protein expression by 47% and 31% respectively, while enriching DNAJB9 mRNA by 1.9-fold after Argonaute2 microribonucleoprotein immunoprecipitation. These effects were mediated through p38 mitogen-activated protein kinase (MAPK). Ischemia-reperfusion (I/R) injury in EC-specific miR-409-3p knockout (KO) mice (miR-409ECKO) fed a high-fat, high-sucrose diet increased isolectin B4 (53.3%), CD31 (56%), and DNAJB9 (41.5%). The left ventricular ejection fraction (EF) was improved by 28%, and the infarct area was decreased by 33.8% in miR-409ECKO compared with control mice. These findings support an important role of miR-409-3p in the angiogenic EC response to myocardial ischemia.

ErbB4 localization to cardiac myocyte nuclei, and its role in myocyte DNA damage response.

The intracellular domain of ErbB4 receptor tyrosine kinase is known to translocate to the nucleus of cells where it can regulate p53 transcriptional activity. The purpose of this study was to examine whether ErbB4 can localize to the nucleus of adult rat ventricular myocytes (ARVM), and regulate p53 in these cells. We demonstrate that ErbB4 does locate to the nucleus of cardiac myocytes as a full-length protein, although nuclear location occurs as a full-length protein that does not require Protein Kinase C or γ-secretase activity. Consistent with this we found that only the non-cleavable JM-b isoform of ErbB4 is expressed in ARVM. Doxorubicin was used to examine ErbB4 role in regulation of a DNA damage response in ARVM. Doxorubicin induced p53 and p21 was suppressed by treatment with AG1478, an EGFR and ErbB4 kinase inhibitor, or suppression of ErbB4 expression with small interfering RNA. Thus ErbB4 localizes to the nucleus as a full-length protein, and plays a role in the DNA damage response induced by doxorubicin in cardiac myocytes.

Role of Kruppel-like factors in leukocyte development, function, and disease.

The Krüppel-like transcription factor (KLF) family participates in diverse aspects of cellular growth, development, differentiation, and activation. Recently, several groups have identified new connections between the function of these factors and leukocyte responses in health and disease. Gene targeting of individual KLFs in mice has uncovered novel and unexpected physiologic roles among myeloid and lymphocyte cell lineage maturation, particularly in the bone marrow niche and blood. In addition, several KLF family members are downstream targets of stimuli and signaling pathways critical to T-cell trafficking, T regulatory cell differentiation or suppressor function, monocyte/macrophage activation or renewal, and B memory cell maturation or activation. Indeed, KLFs have been implicated in subtypes of leukemia, lymphoma, autoimmunity, and in acute and chronic inflammatory disease states, such as atherosclerosis, diabetes, and airway inflammation, raising the possibility that KLFs and their upstream signals are of therapeutic interest. This review focuses on the relevant literature of Krüppel-like factors in leukocyte biology and their implications in clinical settings.

Kruppel-like factor KLF10 targets transforming growth factor-beta1 to regulate CD4(+)CD25(-) T cells and T regulatory cells.

CD4(+)CD25(+) regulatory T cells (T regs) play a major role in the maintenance of self-tolerance and immune suppression, although the mechanisms controlling T reg development and suppressor function remain incompletely understood. Herein, we provide evidence that Kruppel-like factor 10 (KLF10/TIEG1) constitutes an important regulator of T regulatory cell suppressor function and CD4(+)CD25(-) T cell activation through distinct mechanisms involving transforming growth factor (TGF)-beta1 and Foxp3. KLF10 overexpressing CD4(+)CD25(-) T cells induced both TGF-beta1 and Foxp3 expression, an effect associated with reduced T-Bet (Th1 marker) and Gata3 (Th2 marker) mRNA expression. Consistently, KLF10(-/-) CD4(+)CD25(-) T cells have enhanced differentiation along both Th1 and Th2 pathways and elaborate higher levels of Th1 and Th2 cytokines. Furthermore, KLF10(-/-) CD4(+)CD25(-) T cell effectors cannot be appropriately suppressed by wild-type T regs. Surprisingly, KLF10(-/-) T reg cells have reduced suppressor function, independent of Foxp3 expression, with decreased expression and elaboration of TGF-beta1, an effect completely rescued by exogenous treatment with TGF-beta1. Mechanistic studies demonstrate that in response to TGF-beta1, KLF10 can transactivate both TGF-beta1 and Foxp3 promoters, implicating KLF10 in a positive feedback loop that may promote cell-intrinsic control of T cell activation. Finally, KLF10(-/-) CD4(+)CD25(-) T cells promoted atherosclerosis by approximately 2-fold in ApoE(-/-)/scid/scid mice with increased leukocyte accumulation and peripheral pro-inflammatory cytokines. Thus, KLF10 is a critical regulator in the transcriptional network controlling TGF-beta1 in both CD4(+)CD25(-) T cells and T regs and plays an important role in regulating atherosclerotic lesion formation in mice.

Computational Analysis of Targeting SARS-CoV-2, Viral Entry Proteins ACE2 and TMPRSS2, and Interferon Genes by Host MicroRNAs.

Rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for coronavirus disease 2019 (COVID-19), has led to a global pandemic, failures of local health care systems, and global economic recession. MicroRNAs (miRNAs) have recently emerged as important regulators of viral pathogenesis, particularly among RNA viruses, but the impact of host miRNAs on SARS-CoV-2 infectivity remains unknown. In this study, we utilize the combination of powerful bioinformatic prediction algorithms and miRNA profiling to predict endogenous host miRNAs that may play important roles in regulating SARS-CoV-2 infectivity. We provide a collection of high-probability miRNA binding sites within the SARS-CoV-2 genome as well as within mRNA transcripts of critical viral entry proteins ACE2 and TMPRSS2 and their upstream modulators, the interferons (IFN). By utilizing miRNA profiling datasets of SARS-CoV-2-resistant and -susceptible cell lines, we verify the biological plausibility of the predicted miRNA-target RNA interactions. Finally, we utilize miRNA profiling of SARS-CoV-2-infected cells to identify predicted miRNAs that are differentially regulated in infected cells. In particular, we identify predicted miRNA binders to SARS-CoV-2 ORFs (miR-23a (1ab), miR-29a, -29c (1ab, N), miR-151a, -151b (S), miR-4707-3p (S), miR-298 (5'-UTR), miR-7851-3p (5'-UTR), miR-8075 (5'-UTR)), ACE2 3'-UTR (miR-9-5p, miR-218-5p), TMPRSS2 3'-UTR (let-7d-5p, -7e-5p, miR-494-3p, miR-382-3p, miR-181c-5p), and IFN-α 3'-UTR (miR-361-5p, miR-410-3p). Overall, this study provides insight into potential novel regulatory mechanisms of SARS-CoV-2 by host miRNAs and lays the foundation for future investigation of these miRNAs as potential therapeutic targets or biomarkers.

Palmitate alters neuregulin signaling and biology in cardiac myocytes.

The saturated fatty acid palmitate alters normal cell function via disruption of cell signaling, and this effect has been implicated in the end-organ damage associated with dyslipidemia. Neuregulin-1beta (NRG-1beta) is a growth and survival factor in cardiac myocytes. We tested the hypothesis that palmitate alters NRG-1beta signaling and biology in isolated neonatal rat cardiac myocytes. Palmitate treatment inhibited NRG-1beta activation of the PI3-kinase/Akt pathway in myocytes. We found that the pro-apoptotic activity of palmitate was increased by NRG-1beta treatment. The effects of palmitate on NRG-1beta signaling and survival were reversed by the mono-unsaturated fatty acid oleate. Under control conditions NRG-1beta decreases p53 expression in myocytes. In the presence of palmitate, NRG-1beta caused an increase in p53 expression, bax multimer formation, concurrent with degradation of mdm2, a negative regulator of p53. Thus in the presence of palmitate NRG-1beta activates pro-apoptotic, rather than pro-survival signaling in cardiac myocytes.