Estradiol-driven metabolism in transwomen associates with reduced circulating extracellular vesicle microRNA-224/452.

OBJECTIVE: Sex steroid hormones like estrogens have a key role in the regulation of energy homeostasis and metabolism. In transwomen, gender-affirming hormone therapy like estradiol (in combination with antiandrogenic compounds) could affect metabolism as well. Given that the underlying pathophysiological mechanisms are not fully understood, this study assessed circulating estradiol-driven microRNAs (miRs) in transwomen and their regulation of genes involved in metabolism in mice. METHODS: Following plasma miR-sequencing (seq) in a transwomen discovery (n = 20) and validation cohort (n = 30), we identified miR-224 and miR-452. Subsequent systemic silencing of these miRs in male C57Bl/6 J mice (n = 10) was followed by RNA-seq-based gene expression analysis of brown and white adipose tissue in conjunction with mechanistic studies in cultured adipocytes. RESULTS: Estradiol in transwomen lowered plasma miR-224 and -452 carried in extracellular vesicles (EVs) while their systemic silencing in mice and cultured adipocytes increased lipogenesis (white adipose) but reduced glucose uptake and mitochondrial respiration (brown adipose). In white and brown adipose tissue, differentially expressed (miR target) genes are associated with lipogenesis (white adipose) and mitochondrial respiration and glucose uptake (brown adipose). CONCLUSION: This study identified an estradiol-drive post-transcriptional network that could potentially offer a mechanistic understanding of metabolism following gender-affirming estradiol therapy.

Prediction Power on Cardiovascular Disease of Neuroimmune Guidance Cues Expression by Peripheral Blood Monocytes Determined by Machine-Learning Methods.

Atherosclerosis is the underlying pathology in a major part of cardiovascular disease, the leading cause of mortality in developed countries. The infiltration of monocytes into the vessel walls of large arteries is a key denominator of atherogenesis, making monocytes accountable for the development of atherosclerosis. With the development of high-throughput transcriptome profiling platforms and cytometric methods for circulating cells, it is now feasible to study in-depth the predicted functional change of circulating monocytes reflected by changes of gene expression in certain pathways and correlate the changes to disease outcome. Neuroimmune guidance cues comprise a group of circulating- and cell membrane-associated signaling proteins that are progressively involved in monocyte functions. Here, we employed the CIRCULATING CELLS study cohort to classify cardiovascular disease patients and healthy individuals in relation to their expression of neuroimmune guidance cues in circulating monocytes. To cope with the complexity of human datasets featured by noisy data, nonlinearity and multidimensionality, we assessed various machine-learning methods. Of these, the linear discriminant analysis, Naïve Bayesian model and stochastic gradient boost model yielded perfect or near-perfect sensibility and specificity and revealed that expression levels of the neuroimmune guidance cues SEMA6B, SEMA6D and EPHA2 in circulating monocytes were of predictive values for cardiovascular disease outcome.

Circulating Long Noncoding RNA LNC-EPHA6 Associates with Acute Rejection after Kidney Transplantation.

Acute rejection (AR) of a kidney graft in renal transplant recipients is associated with microvascular injury in graft dysfunction and, ultimately, graft failure. Circulating long noncoding RNAs (lncRNAs) may be suitable markers for vascular injury in the context of AR. Here, we first investigated the effect of AR after kidney transplantation on local vascular integrity and demonstrated that the capillary density markedly decreased in AR kidney biopsies compared to pre-transplant biopsies. Subsequently, we assessed the circulating levels of four lncRNAs (LNC-RPS24, LNC-EPHA6, MALAT1, and LIPCAR), that were previously demonstrated to associate with vascular injury in a cohort of kidney recipients with a stable kidney transplant function (n = 32) and recipients with AR (n = 15). The latter were followed longitudinally six and 12 months after rejection. We found higher levels of circulating LNC-EPHA6 during rejection, compared with renal recipients with a stable kidney function (p = 0.017), that normalized one year after AR. In addition, LNC-RPS24, LNC-EPHA6, and LIPCAR levels correlated significantly with the vascular injury marker soluble thrombomodulin. We conclude that AR and microvascular injury are associated with higher levels of circulating LNC-EPHA6, which emphasizes the potential role of lncRNAs as biomarker in the context of AR.

Sex-specific microRNAs in women with diabetes and left ventricular diastolic dysfunction or HFpEF associate with microvascular injury.

Left ventricular diastolic dysfunction (LVDD) and heart failure with preserved ejection fraction (HFpEF) are microcirculation defects following diabetes mellitus (DM). Unrecognized HFpEF is more prevalent in women with diabetes compared to men with diabetes and therefore sex-specific diagnostic strategies are needed. Previously, we demonstrated altered plasma miRs in DM patients with microvascular injury [defined by elevated plasma Angiopoietin-2 (Ang-2) levels]. This study hypothesized the presence of sex-differences in plasma miRs and Ang-2 in diabetic (female) patients with LVDD or HFpEF. After a pilot study, we assessed 16 plasma miRs in patients with LVDD (n = 122), controls (n = 244) and female diabetic patients (n = 10). Subsequently, among these miRs we selected and measured plasma miR-34a, -224 and -452 in diabetic HFpEF patients (n = 53) and controls (n = 52). In LVDD patients, miR-34a associated with Ang-2 levels (R2 0.04, R = 0.21, p = 0.001, 95% CI 0.103-0.312), with plasma levels being diminished in patients with DM, while women with an eGFR < 60 ml/min and LVDD had lower levels of miR-34a, -224 and -452 compared to women without an eGFR < 60 ml/min without LVDD. In diabetic HFpEF women (n = 28), plasma Ang-2 levels and the X-chromosome located miR-224/452 cluster increased compared to men. We conclude that plasma miR-34a, -224 and -452 display an association with the microvascular injury marker Ang-2 and are particularly targeted to women with LVDD or HFpEF.

Diabetic nephropathy alters circulating long noncoding RNA levels that normalize following simultaneous pancreas-kidney transplantation.

Simultaneous pancreas-kidney transplantation (SPKT) replaces kidney function and restores endogenous insulin secretion in patients with diabetic nephropathy (DN). Here, we aimed to identify circulating long noncoding RNAs (lncRNAs) that are associated with DN and vascular injury in the context of SPKT. Based on a pilot study and a literature-based selection of vascular injury-related lncRNAs, we assessed 9 candidate lncRNAs in plasma samples of patients with diabetes mellitus with a kidney function >35 mL/min/1.73 m2 (DM; n = 12), DN (n = 14), SPKT (n = 35), healthy controls (n = 15), and renal transplant recipients (KTx; n = 13). DN patients were also studied longitudinally before and 1, 6, and 12 months after SPKT. Of 9 selected lncRNAs, we found MALAT1, LIPCAR, and LNC-EPHA6 to be higher in DN compared with healthy controls. SPKT caused MALAT1, LIPCAR, and LNC-EPHA6 to normalize to levels of healthy controls, which was confirmed in the longitudinal study. In addition, we observed a strong association between MALAT1, LNC-EPHA6, and LIPCAR and vascular injury marker soluble thrombomodulin and a subset of angiogenic microRNAs (miR-27a, miR-130b, miR-152, and miR-340). We conclude that specific circulating lncRNAs associate with DN-related vascular injury and normalize after SPKT, suggesting that lncRNAs may provide a promising novel monitoring strategy for vascular integrity in the context of SPKT.

Diabetic Nephropathy Alters the Distribution of Circulating Angiogenic MicroRNAs Among Extracellular Vesicles, HDL, and Ago-2.

Previously, we identified plasma microRNA (miR) profiles that associate with markers of microvascular injury in patients with diabetic nephropathy (DN). However, miRs circulate in extracellular vesicles (EVs) or in association with HDL or the RNA-binding protein argonaute-2 (Ago-2). Given that the EV- and HDL-mediated miR transfer toward endothelial cells (ECs) regulates cellular quiescence and inflammation, we hypothesized that the distribution of miRs among carriers affects microvascular homeostasis in DN. Therefore, we determined the miR expression in EV, HDL, and Ago-2 fractions isolated from EDTA plasma of healthy control subjects, patients with diabetes mellitus (DM) with or without early DN (estimated glomerular filtration rate [eGFR] >30 mL/min/1.73 m2), and patients with DN (eGFR <30 mL/min/1.73 m2). Consistent with our hypothesis, we observed alterations in miR carrier distribution in plasma of patients with DM and DN compared with healthy control subjects. Both miR-21 and miR-126 increased in EVs of patients with DN, whereas miR-660 increased in the Ago-2 fraction and miR-132 decreased in the HDL fraction. Moreover, in vitro, differentially expressed miRs improved EC barrier formation (EV-miR-21) and rescued the angiogenic potential (HDL-miR-132) of ECs cultured in serum from patients with DM and DN. In conclusion, miR measurement in EVs, HDL, and Ago-2 may improve the biomarker sensitivity of these miRs for microvascular injury in DN, while carrier-specific miRs can improve endothelial barrier formation (EV-miR-21/126) or exert a proangiogenic response (HDL-miR-132).

Long Non-coding RNAs Rian and Miat Mediate Myofibroblast Formation in Kidney Fibrosis.

There is an increasing prevalence of chronic kidney disease (CKD), which associates with the development of interstitial fibrosis. Pericytes (perivascular fibroblasts) provide a major source of α-SMA-positive myofibroblasts that are responsible for the excessive deposition of extracellular matrix. In order to identify pericyte long non-coding RNAs (lncRNAs) that could serve as a target to decrease myofibroblast formation and counteract the progression of kidney fibrosis we employed two models of experimental kidney injury, one focused on kidney fibrosis (unilateral ureteral obstruction; UUO), and one focused on acute kidney injury that yields kidney fibrosis in the longer term (unilateral ischemia-reperfusion injury; IRI). This was performed in FoxD1-GC;tdTomato stromal cell reporter mice that allowed pericyte fate tracing. Tomato red-positive FoxD1-derivative cells of control and injured kidneys were FACS-sorted and used for lncRNA and mRNA profiling yielding a distinctive transcriptional signature of pericytes and myofibroblasts with 244 and 586 differentially expressed lncRNAs (>twofold, P < 0.05), in the UUO and IRI models, respectively. Next, we selected two differentially expressed and conserved lncRNAs, Rian (RNA imprinted and accumulated in nucleus) and Miat (Myocardial infarction associated transcript), and explored their potential regulatory role in myofibroblast formation through knockdown of their function with gapmers. While Miat was upregulated in myofibroblasts of UUO and IRI in mice, gapmer silencing of Miat attenuated myofibroblast formation as evidenced by decreased expression of α-SMA, col1α1, SMAD2, and SMAD3, as well as decreased α-SMA and pro-collagen-1α1 protein levels. In contrast, silencing Rian, which was found to be downregulated in kidney myofibroblast after IRI and UUO, resulted in increased myofibroblast formation. In addition, we found microRNAs that were previously linked to Miat (miR-150) and Rian (14q32 miRNA cluster), to be dysregulated in the FoxD1-derivative cells, suggesting a possible interaction between miRNAs and these lncRNAs in myofibroblast formation. Taken together, lncRNAs play a regulatory role in myofibroblast formation, possibly through interacting with miRNA regulation, implicating that understanding their biology and their modulation may have the potential to counteract the development of renal fibrosis.

Neutral endopeptidase inhibitors blunt kidney fibrosis by reducing myofibroblast formation.

Kidney fibrosis is the common pathophysiological mechanism in end-stage renal disease characterized by excessive accumulation of myofibroblast-derived extracellular matrix. Natriuretic peptides have been demonstrated to have cyclic guanosine monophosphate (cGMP)-dependent anti-fibrotic properties likely due to interference with pro-fibrotic tissue growth factor ß (TGF-ß) signaling. However, in vivo, natriuretic peptides are rapidly degraded by neutral endopeptidases (NEP). In a unilateral ureteral obstruction (UUO) mouse model for kidney fibrosis we assessed the anti-fibrotic effects of SOL1, an orally active compound that inhibits NEP and endothelin-converting enzyme (ECE). Mice (n=10 per group) subjected to UUO were treated for 1 week with either solvent, NEP-/ECE-inhibitor SOL1 (two doses), reference NEP-inhibitor candoxatril or the angiotensin II receptor type 1 (AT1)-antagonist losartan. While NEP-inhibitors had no significant effect on blood pressure, they did increase urinary cGMP levels as well as endothelin-1 (ET-1) levels. Immunohistochemical staining revealed a marked decrease in renal collagen (∼55% reduction, P<0.05) and α-smooth muscle actin (α-SMA; ∼40% reduction, P<0.05). Moreover, the number of α-SMA positive cells in the kidneys of SOL1-treated groups inversely correlated with cGMP levels consistent with a NEP-dependent anti-fibrotic effect. To dissect the molecular mechanisms associated with the anti-fibrotic effects of NEP inhibition, we performed a 'deep serial analysis of gene expression (Deep SAGE)' transcriptome and targeted metabolomics analysis of total kidneys of all treatment groups. Pathway analyses linked increased cGMP and ET-1 levels with decreased nuclear receptor signaling (peroxisome proliferator-activated receptor [PPAR] and liver X receptor/retinoid X receptor [LXR/RXR] signaling) and actin cytoskeleton organization. Taken together, although our transcriptome and metabolome data indicate metabolic dysregulation, our data support the therapeutic potential of NEP inhibition in the treatment of kidney fibrosis via cGMP elevation and reduced myofibroblast formation.

Acute Rejection After Kidney Transplantation Associates With Circulating MicroRNAs and Vascular Injury.

BACKGROUND: Acute rejection (AR) of kidney transplants is associated with the loss of endothelial integrity, microvascular rarefaction and, ultimately, graft dysfunction. Circulating angiogenic microRNAs (miRNAs) may serve as markers for microvascular injury. Here, we investigated the short- and long-term effects of AR after kidney transplantation on systemic vascular injury and the associated circulating miRNA profile. METHODS: Systemic vascular injury was determined by measuring capillary tortuosity and density within the oral mucosa as well as by assessing circulating levels of angiopoietin-2/angiopoietin-1 ratio, vascular endothelial growth factor and soluble thrombomodulin. After a pilot study, we selected 48 miRNAs to assess the AR- and microvascular injury associated circulating miRNAs. RESULTS: In stable transplant recipients (n = 25) and patients with AR (n = 13), which were also studied longitudinally (1, 6, and 12 months post-AR), we found an AR-associated increase in markers of systemic vascular injury, of which vascular endothelial growth factor and soluble thrombomodulin normalized within 1 year after AR. Of the 48 selected miRNAs, 8 were either decreased (miR-135a, miR-199a-3p, and miR-15a) or increased (miR-17, miR-140-3p, miR-130b, miR-122 and miR-192) in AR. Of these, miR-130b, miR-199a, and miR-192 associated with markers of vascular injury, whereas miR-140-3p, miR-130b, miR-122, and miR-192 normalized within 1 year after AR. CONCLUSIONS: AR after kidney transplantation is characterized by systemic microvascular injury and associates with specific circulating miRNA levels.

Promoting Tropoelastin Expression in Arterial and Venous Vascular Smooth Muscle Cells and Fibroblasts for Vascular Tissue Engineering.

Elastin, critical for its structural and regulatory functions, is a missing link in vascular tissue engineering. Several elastin-inducting compounds have previously been reported, but their relative efficiency in promoting elastogenesis by adult arterial and venous vascular smooth muscle cells (VSMCs) and fibroblasts, four main vascular and elastogenic cells, has not been described. In addition to elasto-inductive substances, microRNA-29a was recently established as a potent post-transcriptional inhibitor of elastogenesis. Here, we explored if stimulating positive regulators or blocking inhibitors of elastogenesis could maximize elastin production. We tested whether the elasto-inducing compounds IGF-1, TGF-ß1, and minoxidil could indeed augment elastin production, and whether microRNA-29a antagonism could block elastin production in adult arterial and venous fibroblasts and VSMCs. The effects on elastin, lysyl oxidase, and fibrillin-1 mRNA expression levels and tropoelastin protein were determined. IGF-1 and minoxidil exerted little effect on tropoelastin mRNA expression levels in all cell types, while TGF-ß1 predominantly enhanced mRNA tropoelastin levels, but this mRNA increase did not impact tropoelastin protein abundance. In contrast, microRNA29a inhibition resulted in the upregulation of tropoelastin mRNA in all cell types, but most pronounced in venous VSMCs. Importantly, microRNA-29a-antagonism also enhanced lysyl oxidase and fibrillin-1 mRNA expression, and revealed a dose-dependent increase in tropoelastin protein expression in venous VSMCs. Our studies suggest that the elastogenic potential of microRNA-29a inhibition in vascular cells is superior to that of established elastin-stimulating compounds IGF-1, TGF-ß1, and minoxidil. Thus, microRNA-29a antagonism could serve as an attractive means of enhancing elastin synthesis in tissue-engineered blood vessels.

Silencing of microRNA-132 reduces renal fibrosis by selectively inhibiting myofibroblast proliferation.

Chronic kidney disease is associated with progressive renal fibrosis, where perivascular cells give rise to the majority of α-smooth muscle actin (α-SMA) positive myofibroblasts. Here we sought to identify pericytic miRNAs that could serve as a target to decrease myofibroblast formation. Kidney fibrosis was induced in FoxD1-GC;Z/Red-mice by unilateral ureteral obstruction followed by FACS sorting of dsRed-positive FoxD1-derivative cells and miRNA profiling. MiR-132 selectively increased 21-fold during pericyte-to-myofibroblast formation, whereas miR-132 was only 2.5-fold up in total kidney lysates (both in obstructive and ischemia-reperfusion injury). MiR-132 silencing during obstruction decreased collagen deposition (35%) and tubular apoptosis. Immunohistochemistry, Western blot, and qRT-PCR confirmed a similar decrease in interstitial α-SMA(+) cells. Pathway analysis identified a rate-limiting role for miR-132 in myofibroblast proliferation that was confirmed in vitro. Indeed, antagomir-132-treated mice displayed a reduction in the number of proliferating Ki67(+) interstitial myofibroblasts. Interestingly, this was selective for the interstitial compartment and did not impair the reparative proliferation of tubular epithelial cells, as evidenced by an increase in Ki67(+) epithelial cells, as well as increased phospho-RB1, Cyclin-A and decreased RASA1, p21 levels in kidney lysates. Additional pathway and gene expression analyses suggest miR-132 coordinately regulates genes involved in TGF-ß signaling (Smad2/Smad3), STAT3/ERK pathways, and cell proliferation (Foxo3/p300). Thus, silencing miR-132 counteracts the progression of renal fibrosis by selectively decreasing myofibroblast proliferation and could potentially serve as a novel antifibrotic therapy.

Quaking promotes monocyte differentiation into pro-atherogenic macrophages by controlling pre-mRNA splicing and gene expression.

A hallmark of inflammatory diseases is the excessive recruitment and influx of monocytes to sites of tissue damage and their ensuing differentiation into macrophages. Numerous stimuli are known to induce transcriptional changes associated with macrophage phenotype, but posttranscriptional control of human macrophage differentiation is less well understood. Here we show that expression levels of the RNA-binding protein Quaking (QKI) are low in monocytes and early human atherosclerotic lesions, but are abundant in macrophages of advanced plaques. Depletion of QKI protein impairs monocyte adhesion, migration, differentiation into macrophages and foam cell formation in vitro and in vivo. RNA-seq and microarray analysis of human monocyte and macrophage transcriptomes, including those of a unique QKI haploinsufficient patient, reveal striking changes in QKI-dependent messenger RNA levels and splicing of RNA transcripts. The biological importance of these transcripts and requirement for QKI during differentiation illustrates a central role for QKI in posttranscriptionally guiding macrophage identity and function.

Development and evaluation of in vivo tissue engineered blood vessels in a porcine model.

BACKGROUND: There's a large clinical need for novel vascular grafts. Tissue engineered blood vessels (TEBVs) have great potential to improve the outcome of vascular grafting procedures. Here, we present a novel approach to generate autologous TEBV in vivo. Polymer rods were engineered and implanted, evoking an inflammatory response that culminates in encapsulation by a fibrocellular capsule. We hypothesized that, after extrusion of the rod, the fibrocellular capsule differentiates into an adequate vascular conduit once grafted into the vasculature. METHODS AND RESULTS: Rods were implanted subcutaneously in pigs. After 4 weeks, rods with tissue capsules grown around it were harvested. Tissue capsules were grafted bilaterally as carotid artery interposition. One and 4-week patency were evaluated by angiography whereupon pigs were sacrificed. Tissue capsules before and after grafting were evaluated on tissue remodeling using immunohistochemistry, RNA profiling and mechanical testing. Rods were encapsulated by thick, well-vascularized tissue capsules, composed of circumferentially aligned fibroblasts, collagen and few leukocytes, with adequate mechanical strength. Patency was 100% after 1 week and 87.5% after 4 weeks. After grafting, tissue capsules remodeled towards a vascular phenotype. Gene profiles of TEBVs gained more similarity with carotid artery. Wall thickness and αSMA-positive area significantly increased. Interestingly, a substantial portion of (myo)fibroblasts present before grafting expressed smooth muscle cell markers. While leukocytes were hardly present anymore, the lumen was largely covered with endothelial cells. Burst pressure remained stable after grafting. CONCLUSIONS: Autologous TEBVs were created in vivo with sufficient mechanical strength enabling vascular grafting. Grafts differentiated towards a vascular phenotype upon grafting.

Aspirin treatment hampers the use of plasma microRNA-126 as a biomarker for the progression of vascular disease.

AIMS: MicroRNA-126 (miR-126) facilitates angiogenesis and regulates endothelial cell function. Recent data suggest that miR-126 can serve as a biomarker for vascular disease. Although endothelial cells are enriched for miR-126, platelets also contain miR-126. In this paper, we investigated the contribution of platelets to the pool of miR-126 in plasma of patients with type 2 diabetes (DM2) and how this is affected by aspirin. METHODS AND RESULTS: In vitro platelet activation resulted in the transfer of miR-126 from the platelet to the plasma compartment, which was prevented by aspirin. In vivo platelet activation, monitored in patients with DM2 by measuring soluble P-selectin, correlated directly with circulating levels of miR-126. The administration of aspirin resulted both in platelet inhibition and concomitantly reduced circulating levels of platelet-derived microRNAs including miR-126. CONCLUSION: Platelets are a major source of circulating miR-126. Consequently, in patho-physiological conditions associated with platelet activation, such as diabetes type 2, the administration of aspirin may lead to reduced levels of circulating miR-126. Thus, the use of platelet inhibitors should be taken into account when using plasma levels of miR-126 as a biomarker.

MicroRNA-155 functions as a negative regulator of RhoA signaling in TGF-ß-induced endothelial to mesenchymal transition.

Endothelial to mesenchymal transition (EndoMT) has been proposed to be involved in the loss of microvascular capillaries in the pathophysiology of fibrosis and organ failure. In EndoMT, endothelial cells (EC) undergo a mesenchymal transition associated with the loss of cell-cell contacts and the acquisition of a synthetic, contractile phenotype. Here, we sought to identify microRNAs (miRNAs) that could play a central role in regulating EndoMT. In a TGF-ß dependent in vitro model for EndoMT, we identified miRNAs that were differentially expressed in normoxic and hypoxic conditions. These studies identified miR-155 to be significantly upregulated in EndoMT, an effect that was enhanced under hypoxia, which further augments EndoMT. Silencing of miR-155 directly increased RhoA expression and activity in endothelial cells and affected phosphorylation of downstream LIMK. In contrast, overexpression of miR-155 counteracted RhoA function. Using a selective Rho kinase inhibitor, we could partly suppress EndoMT, strengthening the notion that RhoA plays a central role in EndoMT. Forced overexpression of miR-155 completely suppressed EndoMT, as evidenced by the maintenance of EC characteristics and blocking the acquisition of a mesenchymal phenotype, as compared to control cells. Our data demonstrate that miRNA-155 functions as a negative regulator of RhoA signaling in TGF-ß-induced endothelial to mesenchymal transition.

Antagomir-mediated silencing of endothelial cell specific microRNA-126 impairs ischemia-induced angiogenesis.

MicroRNAs are negative regulators of gene expression that play a key role in cell-type specific differentiation and modulation of cell function and have been proposed to be involved in neovascularization. Previously, using an extensive cloning and sequencing approach, we identified miR-126 to be specifically and highly expressed in human endothelial cells (EC). Here, we demonstrate EC-specific expression of miR-126 in capillaries and the larger vessels in vivo. We therefore explored the potential role of miR-126 in arteriogenesis and angiogenesis. Using miR-reporter constructs, we show that miR-126 is functionally active in EC in vitro and that it could be specifically repressed using antagomirs specifically targeting miR-126. To study the consequences of miR-126 silencing on vascular regeneration, mice were injected with a single dose of antagomir-126 or a control 'scramblemir' and exposed to ischemia of the left hindlimb by ligation of the femoral artery. Although miR-126 was effectively silenced in mice treated with a single, high dose (HD) of antagomir-126, laser Doppler perfusion imaging did not show effects on blood flow recovery. In contrast, quantification of the capillary density in the gastrocnemius muscle revealed that mice treated with a HD of antagomir-126 had a markedly reduced angiogenic response. Aortic explant cultures of the mice confirmed the role of miR-126 in angiogenesis. Our data demonstrate a facilitary function for miR-126 in ischemia-induced angiogenesis and show the efficacy and specificity of antagomir-induced silencing of EC-specific microRNAs in vivo.

Activation of the lectin pathway in murine lupus nephritis.

In systemic lupus erythematosus (SLE), hypocomplementaemia and complement deposition have been described both in man and in experimental models. A major involvement of the classical pathway of complement activation has been demonstrated in this disease, however relatively little is known about the involvement of the lectin pathway. Therefore in the present study we have analyzed the activity of all three pathways of complement activation in murine models of SLE. In the mouse, MBL is expressed in two forms, namely MBL-A and MBL-C. In the present study young and old MRL-lpr and control MRL+/+ mice were compared for the levels of complement activity with specific attention for the lectin pathway. It was found that upon aging of both MRL-lpr and MRL+/+ mice, a marked decrease in the activity of the classical pathway (CP) occurs. Levels of alternative pathway (AP) and lectin pathway (LP) activity remain unchanged. Key-molecules of these pathways, C1q, C3, MBL-A and MBL-C were analyzed and were all found to be decreased in aged mice of both strains. The levels of MBL-A and MBL-C showed a high degree of correlation and decreased equally. In aged MRL-lpr mice in which autoimmunity is most pronounced, we observed high autoantibody titers and strong deposition of glomerular immune complexes in association with deposition of C1q, C3, MBL-A and MBL-C. In conclusion, these data suggest that in addition to the classical pathway and the alternative pathway also the lectin pathway of complement activation is involved in murine lupus nephritis.

Anti-C1q autoantibodies deposit in glomeruli but are only pathogenic in combination with glomerular C1q-containing immune complexes.

Anti-C1q autoantibodies are present in sera of patients with several autoimmune diseases, including systemic lupus erythematosus (SLE). Strikingly, in SLE the presence of anti-C1q is associated with the occurrence of nephritis. We have generated mouse anti-mouse C1q mAb's and used murine models to investigate whether anti-C1q autoantibodies actually contribute to renal pathology in glomerular immune complex disease. Administration of anti-C1q mAb JL-1, which recognizes the collagen-like region of C1q, resulted in glomerular deposition of C1q and anti-C1q autoantibodies and mild granulocyte influx, but no overt renal damage. However, combination of JL-1 with a subnephritogenic dose of C1q-fixing anti-glomerular basement membrane (anti-GBM) antibodies enhanced renal damage characterized by persistently increased levels of infiltrating granulocytes, major histological changes, and increased albuminuria. This was not observed when a non-C1q-fixing anti-GBM preparation was used. Experiments with different knockout mice showed that renal damage was dependent not only on glomerular C1q and complement activation but also on Fcgamma receptors. In conclusion, anti-C1q autoantibodies deposit in glomeruli together with C1q but induce overt renal disease only in the context of glomerular immune complex disease. This provides an explanation why anti-C1q antibodies are especially pathogenic in patients with SLE.