miR-467 regulates inflammation and blood insulin and glucose.

Obesity is associated with inflammation and insulin resistance (IR), but the regulation of insulin sensitivity (IS) and connections between IS and inflammation remain unclear. We investigated the role of miR-467a-5p, a miRNA induced by hyperglycaemia, in regulating inflammation and blood glucose handling. We previously demonstrated that miR-467a-5p is induced by hyperglycaemia and inhibits the production of thrombospondin-1 (TSP-1), a protein implicated in regulating inflammation. To investigate the role of miR-467 in blood glucose handling and tissue inflammation, WT C57BL/6 mice were fed chow or Western diet from 5 to 32 weeks of age and injected weekly with miR-467a-5p antagonist. Inhibiting miR-467a-5p resulted in 47% increase in macrophage infiltration and increased Il6 levels in adipose tissue, higher plasma insulin levels (98 ng/mL vs 63 ng/mL), and 17% decrease in glucose clearance without increase in weight or HDL/LDL. The antagonist effect was lost in mice on Western diet. Mice lacking TSP-1 lost some but not all of the miR-467 effects, suggesting Thbs1 (and other unknown transcripts) are targeted by miR-467 to regulate inflammation. miR-467a-5p provides a physiological feedback when blood glucose is elevated to avoid inflammation and increased blood glucose and insulin levels, which may prevent IR.

Thrombospondin-4 mediates hyperglycemia- and TGF-beta-induced inflammation in breast cancer.

Inflammation drives the growth of tumors and is an important predictor of cancer aggressiveness. CD68, a marker of tumor-associated macrophages (TAM), is routinely used to aid in prognosis and treatment choices for breast cancer patients. We report that thrombospondin-4 (TSP-4) mediates breast cancer inflammation and growth in mouse models in response to hyperglycemia and TGF-beta by increasing TAM infiltration and production of inflammatory signals in tumors. Analysis of breast cancers and noncancerous tissue specimens from hyperglycemic patients revealed that levels of TSP-4 and of macrophage marker CD68 are upregulated in diabetic tissues. TSP-4 was colocalized with macrophages in cancer tissues. Bone-marrow-derived macrophages (BMDM) responded to high glucose and TGF-beta by upregulating TSP-4 production and expression, as well as the expression of inflammatory markers. We report a novel function for TSP-4 in breast cancer: regulation of TAM infiltration and inflammation. The results of our study provide new insights into regulation of cancer growth by hyperglycemia and TGF-beta and suggest TSP-4 as a potential therapeutic target.

The P387 thrombospondin-4 variant promotes accumulation of macrophages in atherosclerotic lesions.

Thrombospondin-4 (TSP4) is a pro-angiogenic protein that has been implicated in tissue remodeling and local vascular inflammation. TSP4 and, in particular, its SNP variant, P387 TSP4, have been associated with cardiovascular disease. Macrophages are central to initiation and resolution of inflammation and development of atherosclerotic lesions, but the effects of the P387 TSP4 on macrophages remain essentially unknown. We examined the effects of the P387 TSP4 variant on macrophages in cell culture and in vivo in a murine model of atherosclerosis. Furthermore, the levels and distributions of the two TSP4 variants were assessed in human atherosclerotic arteries. In ApoE-/- /P387-TSP4 knock-in mice, lesions size measured by Oil Red O did not change, but the lesions accumulated more macrophages than lesions bearing A387 TSP4. The levels of inflammatory markers were increased in lesions of ApoE-/- /P387-TSP4 knock-in mice compared to ApoE-/- mice. Lesions in human arteries from individuals carrying the P387 variant had higher levels of TSP4 and higher macrophage accumulation. P387 TSP4 was more active in supporting adhesion of cultured human and mouse macrophages in experiments using recombinant TSP4 variants and in cells derived from P387-TSP4 knock-in mice. TSP4 supports the adhesion of macrophages and their accumulation in atherosclerotic lesions without changing the size of lesions. P387 TSP4 is more active in supporting these pro-inflammatory events in the vascular wall, which may contribute to the increased association of P387 TSP4 with cardiovascular disease.

αMß2 Is Antiatherogenic in Female but Not Male Mice.

Atherosclerosis is a complex inflammatory process characterized by monocyte recruitment into the arterial wall, their differentiation into macrophages, and lipid accumulation. Because integrin αMß2 (CD11b/CD18) mediates multiple diverse functions of leukocytes, we examined its role in atherogenesis. αM-/-/ApoE-/- and ApoE-/- mice were fed a control or high fat diet for 3 or 16 wk to induce atherogenesis. Unexpectedly, αM deficiency accelerated development of atherosclerosis in female but not in male mice. The size of aortic root lesions was 3-4.5-fold larger in female αM-/-/ApoE-/- than in ApoE-/- mice. Monocyte and macrophage content within the lesions was increased 2.5-fold in female αM-/-/ApoE-/- mice due to enhanced proliferation. αMß2 elimination promoted gender-dependent foam cell formation due to enhanced uptake of cholesterol by αM-/-/ApoE-/- macrophages. This difference was attributed to enhanced expression of lipid uptake receptors, CD36 and scavenger receptor A1 (SR-A1), in female mice. Macrophages from female αM-/-/ApoE-/- mice showed dramatically reduced expression of FoxM1 transcription factor and estrogen receptors (ER) α and ß. As their antagonists inhibited the effect of 17ß-estradiol (E2), E2 decreased CD36, SR-A1, and foam cell formation in ApoE-/- macrophages in an ERα- and ERß-dependent manner. However, female αM-/-/ApoE-/- macrophages failed to respond to E2 and maintained elevated CD36, SR-A1, and lipid accumulation. FoxM1 inhibition in ApoE-/- macrophages reduced ERs and enhanced CD36 and SR-A1 expression, whereas FoxM1 overexpression in αM-/-/ApoE-/- macrophages reversed their proatherogenic phenotype. We demonstrate a new, surprising atheroprotective role of αMß2 in female ApoE-/- mice. αMß2 maintains ER expression in macrophages and E2-dependent inhibition of foam cell formation.

Kindlin-2 interacts with endothelial adherens junctions to support vascular barrier integrity.

KEY POINTS: A reduction in Kindlin-2 levels in endothelial cells compromises vascular barrier function. Kindlin-2 is a previously unrecognized component of endothelial adherens junctions. By interacting directly and simultaneously with ß- or γ-catenin and cortical actin filaments, Kindlin-2 stabilizes adherens junctions. The Kindlin-2 binding sites for ß- and γ-catenin reside within its F1 and F3 subdomains. Although Kindlin-2 does not associate directly with tight junctions, its downregulation also destabilizes these junctions. Thus, impairment of both adherens and tight junctions may contribute to enhanced leakiness of vasculature in Kindlin-2+/- mice. ABSTRACT: Endothelial cells (EC) establish a physical barrier between the blood and surrounding tissue. Impairment of this barrier can occur during inflammation, ischaemia or sepsis and cause severe organ dysfunction. Kindlin-2, which is primarily recognized as a focal adhesion protein in EC, was not anticipated to have a role in vascular barrier. We tested the role of Kindlin-2 in regulating vascular integrity using several different approaches to decrease Kindlin-2 levels in EC. Reduced levels of Kindlin-2 in Kindlin-2+/- mice aortic endothelial cells (MAECs) from these mice, and human umbilical ECs (HUVEC) treated with Kindlin-2 siRNA showed enhanced basal and platelet-activating factor (PAF) or lipopolysaccharide-stimulated vascular leakage compared to wild-type (WT) counterparts. PAF preferentially disrupted the Kindlin-2+/- MAECs barrier to BSA and dextran and reduced transendothelial resistance compared to WT cells. Kindlin-2 co-localized and co-immunoprecipitated with vascular endothelial cadherin-based complexes, including ß- and γ-catenin and actin, components of adherens junctions (AJ). Direct interaction of Kindlin-2 with ß- and γ-catenin and actin was demonstrated in co-immunoprecipitation and surface plasmon resonance experiments. In thrombin-stimulated HUVECs, Kindlin-2 and cortical actin dissociated from stable AJs and redistributed to radial actin stress fibres of remodelling focal AJs. The ß- and γ-catenin binding site resides within the F1 and F3 subdomains of Kindlin-2 but not the integrin binding site in F3. These results establish a previously unrecognized and vital role of Kindlin-2 with respect to maintaining the vascular barrier by linking Vascuar endothelial cadherin-based complexes to cortical actin and thereby stabilizing AJ.

Integrin αXß2 is a leukocyte receptor for Candida albicans and is essential for protection against fungal infections.

The opportunistic fungus Candida albicans is one of the leading causes of infections in immunocompromised patients, and innate immunity provides a principal mechanism for protection from the pathogen. In the present work, the role of integrin α(X)ß2 in the pathogenesis of fungal infection was assessed. Both purified α(X)ß2 and α(X)ß2-expressing human epithelial kidney 293 cells recognized and bound to the fungal hyphae of SC5314 strain of C. albicans but not to the yeast form or to hyphae of a strain deficient in the fungal mannoprotein, Pra1. The binding of the integrin to the fungus was inhibited by ß-glucans but not by mannans, implicating a lectin-like activity in recognition but distinct in specificity from that of α(M)ß2. Mice deficient in α(X)ß2 were more prone to systemic infection with the LD50 fungal inoculum decreasing 3-fold in α(X)ß2-deficient mice compared with wild-type mice. After challenging i.v. with 1.5 × 104 cell/g, 60% of control C57BL/6 mice died within 14 d compared with 100% mortality of α(X)ß2-deficient mice within 9 d. Organs taken from α(X)ß2-deficient mice 16 h postinfection revealed a 10-fold increase in fungal invasion into the brain and a 2-fold increase into the liver. These data indicate that α(X)ß2 is important for protection against systemic C. albicans infections and macrophage subsets in the liver, Kupffer cells, and in the brain, microglial cells use α(X)ß2 to control fungal invasion.

Kindlin-3 deficiency leads to impaired erythropoiesis and erythrocyte cytoskeleton.

Kindlin-3 (K3) is critical for the activation of integrin adhesion receptors in hematopoietic cells. In humans and mice, K3 deficiency is associated with impaired immunity and bone development, bleeding, and aberrant erythrocyte shape. To delineate how K3 deficiency (K3KO) contributes to anemia and misshaped erythrocytes, mice deficient in erythroid (K3KO∖EpoR-cre) or myeloid cell K3 (K3KO∖Lyz2cre), knockin mice expressing mutant K3 (Q597W598 to AA) with reduced integrin-activation function (K3KI), and control wild-type (WT) K3 mice were studied. Both K3-deficient strains and K3KI mice showed anemia at baseline, reduced response to erythropoietin stimulation, and compromised recovery after phenylhydrazine (PHZ)-induced hemolytic anemia as compared with K3WT. Erythroid K3KO and K3 (Q597W598 to AA) showed arrested erythroid differentiation at proerythroblast stage, whereas macrophage K3KO showed decreased erythroblast numbers at all developmental stages of terminal erythroid differentiation because of reduced erythroblastic island (EBI) formation attributable to decreased expression and activation of erythroblast integrin α4ß1 and macrophage αVß3. Peripheral blood smears of K3KO∖EpoR-cre mice, but not of the other mouse strains, showed numerous aberrant tear drop-shaped erythrocytes. K3 deficiency in these erythrocytes led to disorganized actin cytoskeleton, reduced deformability, and increased osmotic fragility. Mechanistically, K3 directly interacted with F-actin through an actin-binding site K3-LK48. Taken together, these findings document that erythroid and macrophage K3 are critical contributors to erythropoiesis in an integrin-dependent manner, whereas F-actin binding to K3 maintains the membrane cytoskeletal integrity and erythrocyte biconcave shape. The dual function of K3 in erythrocytes and in EBIs establish an important functional role for K3 in normal erythroid function.

Hyperglycemia-Induced miR-467 Drives Tumor Inflammation and Growth in Breast Cancer.

The tumor microenvironment contains the parenchyma, blood vessels, and infiltrating immune cells, including tumor-associated macrophages (TAMs). TAMs affect the developing tumor and drive cancer inflammation. We used mouse models of hyperglycemia and cancer and specimens from hyperglycemic breast cancer (BC) patients to demonstrate that miR-467 mediates the effects of high blood glucose on cancer inflammation and growth. Hyperglycemic patients have a higher risk of developing breast cancer. We have identified a novel miRNA-dependent pathway activated by hyperglycemia that promotes BC angiogenesis and inflammation supporting BC growth. miR-467 is upregulated in endothelial cells (EC), macrophages, BC cells, and in BC tumors. A target of miR-467, thrombospondin-1 (TSP-1), inhibits angiogenesis and promotes resolution of inflammation. Systemic injections of a miR-467 antagonist in mouse models of hyperglycemia resulted in decreased BC growth (p < 0.001). Tumors from hyperglycemic mice had a two-fold increase in macrophage accumulation compared to normoglycemic controls (p < 0.001), and TAM infiltration was prevented by the miR-467 antagonist (p < 0.001). BC specimens from hyperglycemic patients had increased miR-467 levels, increased angiogenesis, decreased levels of TSP-1, and increased TAM infiltration in malignant breast tissue in hyperglycemic vs. normoglycemic patients (2.17-fold, p = 0.002) and even in normal breast tissue from hyperglycemic patients (2.18-fold increase, p = 0.04). In malignant BC tissue, miR-467 levels were upregulated 258-fold in hyperglycemic patients compared to normoglycemic patients (p < 0.001) and increased 56-fold in adjacent normal tissue (p = 0.008). Our results suggest that miR-467 accelerates tumor growth by inducing angiogenesis and promoting the recruitment of TAMs to drive hyperglycemia-induced cancer inflammation.

Effects of thrombospondin-4 on pro-inflammatory phenotype differentiation and apoptosis in macrophages.

Thrombospondin-4 (TSP-4) attracted renewed attention recently as a result of assignment of new functions to this matricellular protein in cardiovascular, muscular, and nervous systems. We have previously reported that TSP-4 promotes local vascular inflammation in a mouse atherosclerosis model. A common variant of TSP-4, P387-TSP-4, was associated with increased cardiovascular disease risk in human population studies. In a mouse atherosclerosis model, TSP-4 had profound effect on accumulation of macrophages in lesions, which prompted us to examine its effects on macrophages in more detail. We examined the effects of A387-TSP-4 and P387-TSP-4 on mouse macrophages in cell culture and in vivo in the model of LPS-induced peritonitis. In tissues and in cell culture, TSP-4 expression was associated with inflammation: TSP-4 expression was upregulated in peritoneal tissues in LPS-induced peritonitis, and pro-inflammatory signals, INFγ, GM-CSF, and LPS, induced TSP-4 expression in macrophages in vivo and in cell culture. Deficiency in TSP-4 in macrophages from Thbs4-/- mice reduced the expression of pro-inflammatory macrophage markers, suggesting that TSP-4 facilitates macrophage differentiation into a pro-inflammatory phenotype. Expression of TSP-4, especially more active P387-TSP-4, was associated with higher cellular apoptosis. Cultured macrophages displayed increased adhesion to TSP-4 and reduced migration in presence of TSP-4, and these responses were further increased with P387 variant. We concluded that TSP-4 expression in macrophages increases their accumulation in tissues during the acute inflammatory process and supports macrophage differentiation into a pro-inflammatory phenotype. In a model of acute inflammation, TSP-4 supports pro-inflammatory macrophage apoptosis, a response that is closely related to their pro-inflammatory activity and release of pro-inflammatory signals. P387-TSP-4 was found to be the more active form of TSP-4 in all examined functions.