Sumita Sinha

Mice deficient in α-actinin-4 have severe glomerular disease

Dominantly inherited mutations in ACTN4, which encodes α-actinin-4, cause a form of human focal and segmental glomerulosclerosis (FSGS). By homologous recombination in ES cells, we developed a mouse model deficient in Actn4. Mice homozygous for the targeted allele have no detectable α-actinin-4 protein expression. The number of homozygous mice observed was lower than expected under mendelian inheritance. Surviving mice homozygous for the targeted allele show progressive proteinuria, glomerular disease, and typically death by several months of age. Light microscopic analysis shows extensive glomerular disease and proteinaceous casts. Electron microscopic examination shows focal areas of podocyte foot-process effacement in young mice, and diffuse effacement and globally disrupted podocyte morphology in older mice. Despite the widespread distribution of α-actinin-4, histologic examination of mice showed abnormalities only in the kidneys. In contrast to the dominantly inherited human form of ACTN4-associated FSGS, here we show that the absence of α-actinin-4 causes a recessive form of disease in mice. Cell motility, as measured by lymphocyte chemotaxis assays, was increased in the absence of α-actinin-4. We conclude that α-actinin-4 is required for normal glomerular function. We further conclude that the nonsarcomeric forms of α-actinin (α-actinin-1 and α-actinin-4) are not functionally redundant. In addition, these genetic studies demonstrate that the nonsarcomeric α-actinin-4 is involved in the regulation of cell movement.

Kruppel-like Factor 4 Regulates Endothelial Inflammation

The vascular endothelium plays a critical role in vascular homeostasis. Inflammatory cytokines and non-laminar blood flow induce endothelial dysfunction and confer a pro-adhesive and pro-thrombotic phenotype. Therefore, identification of factors that mediate the effects of these stimuli on endothelial function is of considerable interest. Kruppel-like factor 4 expression has been documented in endothelial cells, but a function has not been described. In this communication we describe the expression in vitro and in vivo of Kruppel-like factor 4 in human and mouse endothelial cells. Furthermore, we demonstrate that endothelial Kruppel-like factor 4 is induced by pro-inflammatory stimuli and shear stress. Overexpression of Kruppel-like factor 4 induces expression of multiple anti-inflammatory and anti-thrombotic factors including endothelial nitric-oxide synthase and thrombomodulin, whereas knockdown of Kruppellike factor 4 leads to enhancement of tumor necrosis factor α-induced vascular cell adhesion molecule-1 and tissue factor expression. The functional importance of Kruppel-like factor 4 is verified by demonstrating that Kruppel-like factor 4 expression markedly decreases inflammatory cell adhesion to the endothelial surface and prolongs clotting time under inflammatory states. Kruppel-like factor 4 differentially regulates the promoter activity of pro- and anti-inflammatory genes in a manner consistent with its anti-inflammatory function. These data implicate Kruppel-like factor 4 as a novel regulator of endothelial activation in response to pro-inflammatory stimuli.

2-Aminoadipic acid is a biomarker for diabetes risk

Improvements in metabolite-profiling techniques are providing increased breadth of coverage of the human metabolome and may highlight biomarkers and pathways in common diseases such as diabetes. Using a metabolomics platform that analyzes intermediary organic acids, purines, pyrimidines, and other compounds, we performed a nested case-control study of 188 individuals who developed diabetes and 188 propensity-matched controls from 2,422 normoglycemic participants followed for 12 years in the Framingham Heart Study. The metabolite 2-aminoadipic acid (2-AAA) was most strongly associated with the risk of developing diabetes. Individuals with 2-AAA concentrations in the top quartile had greater than a 4-fold risk of developing diabetes. Levels of 2-AAA were not well correlated with other metabolite biomarkers of diabetes, such as branched chain amino acids and aromatic amino acids, suggesting they report on a distinct pathophysiological pathway. In experimental studies, administration of 2-AAA lowered fasting plasma glucose levels in mice fed both standard chow and high-fat diets. Further, 2-AAA treatment enhanced insulin secretion from a pancreatic β cell line as well as murine and human islets. These data highlight a metabolite not previously associated with diabetes risk that is increased up to 12 years before the onset of overt disease. Our findings suggest that 2-AAA is a marker of diabetes risk and a potential modulator of glucose homeostasis in humans.

Inhibition of Atherogenesis in BLT1-Deficient Mice Reveals a Role for LTB4 and BLT1 in Smooth Muscle Cell Recruitment

Background—It is known that 5-lipoxygenase and its product, leukotriene B4 (LTB4), are highly expressed in several human pathologies, including atherosclerotic plaque. LTB4 signals primarily through its high-affinity G protein-coupled receptor BLT1, which is expressed on specific leukocyte subsets. BLT1 receptor expression and function on other atheroma-associated cell types is unknown. Methods and Results—To directly assess the role of the LTB4-BLT1 pathway in atherogenesis, we bred BLT1−/− mice into the atherosclerosis-susceptible apoE−/− strain. Compound-deficient apoE−/−/Blt1−/− mice fed a Western-type diet had a marked reduction in plaque formation compared with apoE−/− controls. Immunohistochemical analysis of atherosclerotic lesions in compound-deficient mice revealed a striking decrease in smooth muscle cells (SMCs) and significant decreases in macrophages and T cells. We report here novel evidence of the expression and function of BLT1 on vascular SMCs. LTB4 triggered SMC chemotaxis, which was pertussis toxin sensitive in Blt1+/+ SMCs and absent in Blt1−/− cells, suggesting that BLT1 was the dominant receptor mediating effector functions through a G protein-coupled signaling pathway. Furthermore, BLT1 colocalized with SMCs in human atherosclerotic lesions. Conclusions—These new findings extend the role of inducible BLT1 to nonleukocyte populations and suggest an important target for intervention to modulate the response to vascular injury.

Integrin-linked kinase regulates endothelial cell survival and vascular development

ABSTRACT Integrin-linked kinase (ILK) is a phosphoinositide 3-kinase-dependent serine/threonine kinase that interacts with β integrins. Here we show that endothelial cell (EC)-specific deletion of ILK in mice confers placental insufficiency with decreased labyrinthine vascularization, yielding no viable offspring. Deletion of ILK in zebra fish using antisense morpholino oligonucleotides results in marked patterning abnormalities of the vasculature and is similarly lethal. To dissect potential mechanisms responsible for these phenotypes, we performed ex vivo deletion of ILK from purified EC of adult mice. We observed downregulation of the active-conformation of β1 integrins with a striking increase in EC apoptosis associated with activation of caspase 9. There was also reduced phosphorylation of the ILK kinase substrate, Akt. However, phenotypic rescue of ILK-deficient EC by wild-type ILK, but not by a constitutively active mutant of Akt, suggests regulation of EC survival by ILK in an Akt-independent manner. Thus, endothelial ILK plays a critical role in vascular development through integrin-matrix interactions and EC survival. These data have important implications for both physiological and pathological angiogenesis.

α-Actinin-4 Is Required for Normal Podocyte Adhesion

Mutations in the α-actinin-4 gene ACTN4 cause an autosomal dominant human kidney disease. Mice deficient in α-actinin-4 develop a recessive phenotype characterized by kidney failure, proteinuria, glomerulosclerosis, and retraction of glomerular podocyte foot processes. However, the mechanism by which α-actinin-4 deficiency leads to glomerular disease has not been defined. Here, we examined the effect of α-actinin-4 deficiency on the adhesive properties of podocytes in vivo and in a cell culture system. In α-actinin-4-deficient mice, we observed a decrease in the number of podocytes per glomerulus compared with wild-type mice as well as the presence of podocyte markers in the urine. Podocyte cell lines generated from α-actinin-4-deficient mice were less adherent than wild-type cells to glomerular basement membrane (GBM) components collagen IV and laminin 10 and 11. We also observed markedly reduced adhesion of α-actinin-4-deficient podocytes under increasing shear stresses. This adhesion deficit was restored by transfecting cells with α-actinin-4-GFP. We tested the strength of the integrin receptor-mediated linkages to the cytoskeleton by applying force to microbeads bound to integrin using magnetic pulling cytometry. Beads bound toα-actinin-4-deficient podocytes showed greater displacement in response to an applied force than those bound to wild-type cells. Consistent with integrin-dependent α-actinin-4-mediated adhesion, phosphorylation of β1-integrins on α-actinin-4-deficient podocytes is reduced. We rescued the phosphorylation deficit by transfecting α-actinin-4 into α-actinin-4-deficient podocytes. These results suggest that α-actinin-4 interacts with integrins and strengthens the podocyte-GBM interaction thereby stabilizing glomerular architecture and preventing disease.

Kynurenic acid triggers firm arrest of leukocytes to vascular endothelium under flow conditions

Recent studies have demonstrated that kynurenic acid (KYNA), a compound produced endogenously by the interferon-γ-induced degradation of tryptophan by indoleamine 2,3-dioxygenase, activates the previously orphaned G protein-coupled receptor, GPR35. This receptor is expressed in immune tissues, although its potential function in immunomodulation remains to be explored. We determined that GPR35 was most highly expressed on human peripheral monocytes. In an in vitro vascular flow model, KYNA triggered the firm arrest of monocytes to both fibronectin and ICAM-1, via β1 integrin- and β2 integrin-mediated mechanisms, respectively. Incubation of monocytes with pertussis toxin prior to use in flow experiments significantly reduced the KYNA-induced monocyte adhesion, suggesting that adhesion is triggered by a Gi-mediated process. Furthermore, KYNA-triggered adhesion of monocytic cells was reduced by short hairpin RNA-mediated silencing of GPR35. Although GPR35 is expressed at slightly lower levels on neutrophils, KYNA induced firm adhesion of these cells to an ICAM-1-expressing monolayer as well. KYNA also elicited neutrophil shedding of surface L-selectin, another indicator of leukocyte activation. Taken together, these data suggest that KYNA could be an important early mediator of leukocyte recruitment.

Pharmacological Inhibition of a MicroRNA Family in Nonhuman Primates by a Seed-Targeting 8-Mer AntimiR

Long-term treatment of obese, insulin-resistant nonhuman primates with a seed-targeting antimiR oligonucleotide against the microRNA-33 family derepresses hepatic expression of miR-33 targets, increases circulating HDL cholesterol, and has a clean safety profile. Little AntimiR Packs a Double Punch MicroRNAs (miRNAs) are a type of noncoding RNA that are about 22 nucleotides in length and affect a variety of cellular functions, including normal development and metabolism. These RNAs have also been implicated in many different diseases. Targeted inhibition of miRNAs can be achieved with antimiRs—RNA segments with complementary sequences to miRNAs of interest, which can bind and specifically inhibit their target miRNAs. In humans, miRNAs called miR-33a and miR-33b help control the homeostasis of cholesterol and other lipids, which are associated with cardiovascular disease. To inhibit both of these miRNAs at the same time, Rottiers and colleagues created an unusually short antimiR, only 8 nucleic acids in length, which targets the common portion of both miR-33a and miR-33b. They had tested it in mammalian cells and in mice, and now also confirmed that this short antimiR can be used in nonhuman primates. The authors demonstrated that their antimiR is safe in obese, insulin-resistant nonhuman primates, and that it increases high-density lipoprotein cholesterol. Additional studies will be necessary to learn more about the effects of this 8-mer antimiR on different parameters of metabolism, and to determine how it affects clinical outcomes, such as the risk of death from cardiovascular disease. Nevertheless, this work suggests that miRNA-based approaches could be specifically tailored and potentially safe for patient use, providing an alternative to standard pharmaceutical interventions. MicroRNAs (miRNAs) regulate many aspects of human biology. They target mRNAs for translational repression or degradation through base pairing with 3′ untranslated regions, primarily via seed sequences (nucleotides 2 to 8 in the mature miRNA sequence). A number of individual miRNAs and miRNA families share seed sequences and targets, but differ in the sequences outside of the seed. miRNAs have been implicated in the etiology of a wide variety of human diseases and therefore represent promising therapeutic targets. However, potential redundancy of different miRNAs sharing the same seed sequence and the challenge of simultaneously targeting miRNAs that differ significantly in nonseed sequences complicate therapeutic targeting approaches. We recently demonstrated effective inhibition of entire miRNA families using seed-targeting 8-mer locked nucleic acid (LNA)–modified antimiRs in short-term experiments in mammalian cells and in mice. However, the long-term efficacy and safety of this approach in higher organisms, such as humans and nonhuman primates, have not been determined. We show that pharmacological inhibition of the miR-33 family, key regulators of cholesterol/lipid homeostasis, by a subcutaneously delivered 8-mer LNA-modified antimiR in obese and insulin-resistant nonhuman primates results in derepression of miR-33 targets, such as ABCA1, increases circulating high-density lipoprotein cholesterol, and is well tolerated over 108 days of treatment. These findings demonstrate the efficacy and safety of an 8-mer LNA-antimiR against an miRNA family in a nonhuman primate metabolic disease model, suggesting that this could be a feasible approach for therapeutic targeting of miRNA families sharing the same seed sequence in human diseases.

Genome-wide identification of microRNAs regulating cholesterol and triglyceride homeostasis

Genome-wide association studies (GWASs) have linked genes to various pathological traits. However, the potential contribution of regulatory noncoding RNAs, such as microRNAs (miRNAs), to a genetic predisposition to pathological conditions has remained unclear. We leveraged GWAS meta-analysis data from >188,000 individuals to identify 69 miRNAs in physical proximity to single-nucleotide polymorphisms (SNPs) associated with abnormal levels of circulating lipids. Several of these miRNAs (miR-128-1, miR-148a, miR-130b, and miR-301b) control the expression of key proteins involved in cholesterol-lipoprotein trafficking, such as the low-density lipoprotein (LDL) receptor (LDLR) and the ATP-binding cassette A1 (ABCA1) cholesterol transporter. Consistent with human liver expression data and genetic links to abnormal blood lipid levels, overexpression and antisense targeting of miR-128-1 or miR-148a in high-fat diet–fed C57BL/6J and Apoe-null mice resulted in altered hepatic expression of proteins involved in lipid trafficking and metabolism, and in modulated levels of circulating lipoprotein-cholesterol and triglycerides. Taken together, these findings support the notion that altered expression of miRNAs may contribute to abnormal blood lipid levels, predisposing individuals to human cardiometabolic disorders.

Targeted deletion of integrin-linked kinase reveals a role in T-cell chemotaxis and survival.

ABSTRACT Integrin-linked kinase (ILK) is a serine/threonine kinase that is important in cell-matrix interactions and cell signaling. To examine the role of ILK in leukocyte trafficking and survival, we generated T cell-specific ILK knockouts by breeding ILKflox/flox mice to transgenic mice expressing Cre recombinase under control of the Lck proximal promoter. Thymic T cells from Lck-Cre+/ILKflox/flox mice had a marked reduction (>95%) in ILK protein levels. Thymic cellularity was comparable in 3- to 4-week-old mice, but a threefold diminution of thymic T cells became evident by 6 to 8 weeks of age in the T cell-specific ILK knockout mice due to increased cell death of double-positive (DP) T cells. Analysis of peripheral T cells by quantitative PCR and by breeding Lck-Cre+/ILKflox/flox mice to a YFP-transgenic reporter strain demonstrated an approximate 20-fold enrichment of ILK-competent cells, suggesting these cells have a competitive advantage in trafficking to and/or survival in peripheral lymphatic organs. We explored mechanisms related to altered cell trafficking and survival that might explain the decreases in thymic cellularity and enrichment for ILK-competent cells in the spleen and lymph nodes. We observed a >50% reduction in chemotaxis of ILK-deficient T cells to the chemokines CXCL12 (stromal cell-derived factor [SDF]-1α) and CCL19 (macrophage inflammatory protein [MIP]-3β), as well as enhanced apoptosis of ILK-deficient cells upon stress. Signaling studies in ILK-deficient T cells demonstrated diminished phosphorylation of Akt on the activating phosphorylation site, Ser 473, and a concordant decrease in Akt kinase activity following stimulation with the chemokine SDF-1. Rac1 activation was also markedly diminished in ILK-deficient T cells following chemokine stimulation. These data extend the role of ILK to immune-cell trafficking and survival via modulation of Akt- and Rac-dependent substrates, and have implications for cell recruitment in both homeostatic and pathological processes.