VEGF-Induced Expression of miR-17-92 Cluster in Endothelial Cells Is Mediated by ERK/ELK1 Activation and Regulates Angiogenesis.

RATIONALE: Several lines of evidence indicate that the regulation of microRNA (miRNA) levels by different stimuli may contribute to the modulation of stimulus-induced responses. The miR-17-92 cluster has been linked to tumor development and angiogenesis, but its role in vascular endothelial growth factor-induced endothelial cell (EC) functions is unclear and its regulation is unknown. OBJECTIVE: The purpose of this study was to elucidate the mechanism by which VEGF regulates the expression of miR-17-92 cluster in ECs and determine its contribution to the regulation of endothelial angiogenic functions, both in vitro and in vivo. This was done by analyzing the effect of postnatal inactivation of miR-17-92 cluster in the endothelium (miR-17-92 iEC-KO mice) on developmental retinal angiogenesis, VEGF-induced ear angiogenesis, and tumor angiogenesis. METHODS AND RESULTS: Here, we show that Erk/Elk1 activation on VEGF stimulation of ECs is responsible for Elk-1-mediated transcription activation (chromatin immunoprecipitation analysis) of the miR-17-92 cluster. Furthermore, we demonstrate that VEGF-mediated upregulation of the miR-17-92 cluster in vitro is necessary for EC proliferation and angiogenic sprouting. Finally, we provide genetic evidence that miR-17-92 iEC-KO mice have blunted physiological retinal angiogenesis during development and diminished VEGF-induced ear angiogenesis and tumor angiogenesis. Computational analysis and rescue experiments show that PTEN (phosphatase and tensin homolog) is a target of the miR-17-92 cluster and is a crucial mediator of miR-17-92-induced EC proliferation. However, the angiogenic transcriptional program is reduced when miR-17-92 is inhibited. CONCLUSIONS: Taken together, our results indicate that VEGF-induced miR-17-92 cluster expression contributes to the angiogenic switch of ECs and participates in the regulation of angiogenesis.

Endothelial miR-17∼92 cluster negatively regulates arteriogenesis via miRNA-19 repression of WNT signaling.

The contribution of endothelial-derived miR-17∼92 to ischemia-induced arteriogenesis has not been investigated in an in vivo model. In the present study, we demonstrate a critical role for the endothelial-derived miR-17∼92 cluster in shaping physiological and ischemia-triggered arteriogenesis. Endothelial-specific deletion of miR-17∼92 results in an increase in collateral density limbs and hearts and in ischemic limbs compared with control mice, and consequently improves blood flow recovery. Individual cluster components positively or negatively regulate endothelial cell (EC) functions in vitro, and, remarkably, ECs lacking the cluster spontaneously form cords in a manner rescued by miR-17a, -18a, and -19a. Using both in vitro and in vivo analyses, we identified FZD4 and LRP6 as targets of miR-19a/b. Both of these targets were up-regulated in 17∼92 KO ECs compared with control ECs, and both were shown to be targeted by miR-19 using luciferase assays. We demonstrate that miR-19a negatively regulates FZD4, its coreceptor LRP6, and WNT signaling, and that antagonism of miR-19a/b in aged mice improves blood flow recovery after ischemia and reduces repression of these targets. Collectively, these data provide insights into miRNA regulation of arterialization and highlight the importance of vascular WNT signaling in maintaining arterial blood flow.

Dynamin 2 regulation of integrin endocytosis, but not VEGF signaling, is crucial for developmental angiogenesis.

Here we show that dynamin 2 (Dnm2) is essential for angiogenesis in vitro and in vivo. In cultured endothelial cells lacking Dnm2, vascular endothelial growth factor (VEGF) signaling and receptor levels are augmented whereas cell migration and morphogenesis are impaired. Mechanistically, the loss of Dnm2 increases focal adhesion size and the surface levels of multiple integrins and reduces the activation state of ß1 integrin. In vivo, the constitutive or inducible loss of Dnm2 in endothelium impairs branching morphogenesis and promotes the accumulation of ß1 integrin at sites of failed angiogenic sprouting. Collectively, our data show that Dnm2 uncouples VEGF signaling from function and coordinates the endocytic turnover of integrins in a manner that is crucially important for angiogenesis in vitro and in vivo.

eNOS-derived nitric oxide regulates endothelial barrier function through VE-cadherin and Rho GTPases.

Transient disruption of endothelial adherens junctions and cytoskeletal remodeling are responsible for increases in vascular permeability induced by inflammatory stimuli and vascular endothelial growth factor (VEGF). Nitric oxide (NO) produced by endothelial NO synthase (eNOS) is crucial for VEGF-induced changes in permeability in vivo; however, the molecular mechanism by which endogenous NO modulates endothelial permeability is not clear. Here, we show that the lack of eNOS reduces VEGF-induced permeability, an effect mediated by enhanced activation of the Rac GTPase and stabilization of cortical actin. The loss of NO increased the recruitment of the Rac guanine-nucleotide-exchange factor (GEF) TIAM1 to adherens junctions and VE-cadherin (also known as cadherin 5), and reduced Rho activation and stress fiber formation. In addition, NO deficiency reduced VEGF-induced VE-cadherin phosphorylation and impaired the localization, but not the activation, of c-Src to cell junctions. The physiological role of eNOS activation is clear given that VEGF-, histamine- and inflammation-induced vascular permeability is reduced in mice bearing a non-phosphorylatable knock-in mutation of the key eNOS phosphorylation site S1176. Thus, NO is crucial for Rho GTPase-dependent regulation of cytoskeletal architecture leading to reversible changes in vascular permeability.

Morphogenesis of the developing mammary gland: stage-dependent impact of adipocytes.

Mammary gland development is critically dependent on the interactions between the stromal and the epithelial compartments within the gland. These events are under the control of a complex interplay of circulating and locally acting hormones and growth factors. To analyze the temporal and quantitative contributions of stromal adipocytes, we took advantage of the FAT-ATTAC mice (apoptosis through triggered activation of caspase-8), a model of inducible and reversible loss of adipocytes. This loss can be achieved through the induced dimerization of a caspase-8 fusion protein. In the context of female mice, we can achieve ablation of mammary adipocytes relatively selectively without affecting other fat pads. Under these conditions, we find that adipocytes are essential for the formation of the extended network of ducts in the mammary gland during puberty. Beyond their role in development, adipocytes are also essential to maintain the normal alveolar structures that develop during adulthood. Loss of adipose tissue initiated 2 weeks after birth triggers fewer duct branching points and fewer terminal end buds (TEBs) and also triggers changes in proliferation and apoptosis in the epithelium associated with the TEBs. The reduced developmental pace that adipocyte-ablated glands undergo is reversible, as the emergence of new local adipocytes, upon cessation of treatment, enables the ductal epithelium to resume growth. Conversely, loss of local adipocytes initiated at 7 weeks of age resulted in excessive lobulation, indicating that adipocytes are critically involved in maintaining proper architecture and functionality of the mammary epithelium. Collectively, using a unique model of inducible and reversible loss of adipocytes, our observations suggest that adipocytes are required for proper development during puberty and for the maintenance of the ductal architecture in the adult mammary gland.

Targeting G542X CFTR nonsense alleles with ELX-02 restores CFTR function in human-derived intestinal organoids.

BACKGROUND: Promoting full-length protein production is a requisite step to address some of the remaining unmet medical need for those with Cystic Fibrosis (CF) nonsense alleles. ELX-02 promotes read-through of mRNA transcripts bearing nonsense mutations, including the most common CF nonsense allele G542X, in several different preclinical models including human bronchial epithelial cells. Here we evaluate ELX-02 mediated read-through using the CFTR-dependent Forskolin-induced swelling (FIS) assay across a selection of G542X genotype patient derived organoids (PDOs). METHODS: CFTR functional restoration was evaluated in ELX-02 treated G542X homozygous and heterozygous PDOs in the CFTR-dependent FIS assay. CFTR mRNA abundance and integrity were evaluated by qPCR and Nanostring analysis while PDO protein was detected by capillary based size-exclusion chromatography. RESULTS: PDOs homozygous for G542X or heterozygous with a second minimally functional allele had significantly increased CFTR activity with ELX-02 in a dose-dependent fashion across a variety of forskolin induction concentrations. The functional increases are similar to those obtained with tezacaftor/ivacaftor in F508del homozygous PDOs. Increased CFTR C- and B-band protein was observed in accordance with increased function. In addition, ELX-02 treatment of a G542X/G542X PDO results in a 5-fold increase in CFTR mRNA compared with vehicle treated, resulting in normalization of CFTR mRNA as measured via Nanostring. CONCLUSIONS: These data with ELX-02 in PDOs are consistent with previous G542X model evaluations. These results also support the on-going clinical evaluation of ELX-02 as a read-through agent for CF caused by the G542X allele.

Proangiogenic contribution of adiponectin toward mammary tumor growth in vivo.

PURPOSE: Adipocytes represent one of the most abundant constituents of the mammary gland. They are essential for mammary tumor growth and survival. Metabolically, one of the more important fat-derived factors ("adipokines") is adiponectin (APN). Serum concentrations of APN negatively correlate with body mass index and insulin resistance. To explore the association of APN with breast cancer and tumor angiogenesis, we took an in vivo approach aiming to study its role in the mouse mammary tumor virus (MMTV)-polyoma middle T antigen (PyMT) mammary tumor model. EXPERIMENTAL DESIGN: We compared the rates of tumor growth in MMTV-PyMT mice in wild-type and APN-null backgrounds. RESULTS: Histology and micro-positron emission tomography imaging show that the rate of tumor growth is significantly reduced in the absence of APN at early stages. PyMT/APN knockout mice exhibit a reduction in their angiogenic profile resulting in nutrient deprivation of the tumors and tumor-associated cell death. Surprisingly, in more advanced malignant stages of the disease, tumor growth develops more aggressively in mice lacking APN, giving rise to a larger tumor burden, an increase in the mobilization of circulating endothelial progenitor cells, and a gene expression fingerprint indicative of more aggressive tumor cells. CONCLUSIONS: These observations highlight a novel important contribution of APN in mammary tumor development and angiogenesis, indicating that APN has potent angio-mimetic properties in tumor vascularization. However, in tumors deprived of APN, this antiangiogenic stress results in an adaptive response that fuels tumor growth through mobilization of circulating endothelial progenitor cells and the development of mechanisms enabling massive cell proliferation despite a chronically hypoxic microenvironment.

Autologous T-lymphocytes stimulate proliferation of orbital fibroblasts derived from patients with Graves' ophthalmopathy.

PURPOSE: Graves' ophthalmopathy (GO) affects 50% to 60% of patients with Graves' hyperthyroidism, resulting in exophthalmos, periorbital edema, pain, double vision, optic neuropathy, and loss of vision. Fibroblasts are a key autoimmune target in GO and have effector functions that contribute to GO-associated pathologic conditions, including proliferation, production of excess glycosaminoglycans, and fat deposition. GO is also characterized by autoimmune inflammation of orbital connective tissue with mononuclear cell infiltration, including T cells. METHODS: To determine whether autologous T cells can drive proliferation of orbital fibroblasts and thus contribute to GO, a novel reverse autologous mixed-cell reaction (rAMCR) was performed. Fibroblasts cultured from orbital tissue of patients with GO that was removed during orbital decompression surgery were mixed with autologous T cells, and fibroblast proliferation was determined. RESULTS: Autologous T cells stimulated proliferation of orbital fibroblasts. Fibroblasts derived from blepharoplasty fat of two different patients did not proliferate, demonstrating that the effect is specific to cells derived from deep orbital fat. Proliferation was dependent on cell contact and on major histocompatibility complex (MHC) class II and CD40-CD154 (CD40 ligand) signaling. CONCLUSIONS: The results suggest that T cells and orbital fibroblasts participate in an antigen-dependent positive feedback loop in which presentation of autoantigens by fibroblasts via MHC class II and CD40-CD40L signaling results in T-cell activation. These activated T cells stimulate fibroblast proliferation, leading to fibroblast-associated diseases in GO. Thus, therapies that interfere with CD40-CD40L signaling, antigen expression by fibroblasts, or T-cell function may be effective in preventing progression of GO symptoms.

miRNAs as modulators of angiogenesis.

MicroRNAs are highly expressed in endothelial cells, and recent data suggest that they regulate aspects of vascular development and angiogenesis. This study highlights the state of the art in this field and potential therapeutic opportunities. MicroRNAs (miRNAs) represent a family of conserved short (≈22 nt) noncoding single-stranded RNAs that have been identified in plants and animals. They are generated by the sequential processing of the RNA template by the enzymes Drosha and Dicer, and mature miRNAs can regulate the levels of gene expression at the posttranscriptional level. miRNAs participate in a diverse range of regulatory events via regulation of genes involved in the control of processes such as development, differentiation, homeostasis, metabolism, growth, proliferation, and apoptosis. However, rather than functioning as regulatory on-off switches, miRNAs often function to modulate or fine-tune cellular phenotypes. So far, more than 1000 mammalian miRNAs have been identified since the discovery of the first two miRNAs (lin-4 and let-7), and bioinformatics predictions indicate that mammalian miRNAs can regulate ∼30% of all protein-coding genes.

Hypoxia-inducible factor 1alpha induces fibrosis and insulin resistance in white adipose tissue.

Adipose tissue can undergo rapid expansion during times of excess caloric intake. Like a rapidly expanding tumor mass, obese adipose tissue becomes hypoxic due to the inability of the vasculature to keep pace with tissue growth. Consequently, during the early stages of obesity, hypoxic conditions cause an increase in the level of hypoxia-inducible factor 1alpha (HIF1alpha) expression. Using a transgenic model of overexpression of a constitutively active form of HIF1alpha, we determined that HIF1alpha fails to induce the expected proangiogenic response. In contrast, we observed that HIF1alpha initiates adipose tissue fibrosis, with an associated increase in local inflammation. "Trichrome- and picrosirius red-positive streaks," enriched in fibrillar collagens, are a hallmark of adipose tissue suffering from the early stages of hypoxia-induced fibrosis. Lysyl oxidase (LOX) is a transcriptional target of HIF1alpha and acts by cross-linking collagen I and III to form the fibrillar collagen fibers. Inhibition of LOX activity by beta-aminoproprionitrile treatment results in a significant improvement in several metabolic parameters and further reduces local adipose tissue inflammation. Collectively, our observations are consistent with a model in which adipose tissue hypoxia serves as an early upstream initiator for adipose tissue dysfunction by inducing a local state of fibrosis.

Activated human T lymphocytes express cyclooxygenase-2 and produce proadipogenic prostaglandins that drive human orbital fibroblast differentiation to adipocytes.

The differentiation of preadipocyte fibroblasts to adipocytes is a crucial process to many disease states including obesity, cardiovascular, and autoimmune diseases. In Graves' disease, the orbit of the eye can become severely inflamed and infiltrated with T lymphocytes as part of the autoimmune process. The orbital fibroblasts convert to fat-like cells causing the eye to protrude, which is disfiguring and can lead to blindness. Recently, the transcription factor peroxisome proliferator activated receptor (PPAR)-gamma and its natural (15d-PGJ2) and synthetic (thiazolidinedione-type) PPAR-gamma agonists have been shown to be crucial to the in vitro differentiation of preadipocyte fibroblasts to adipocytes. We show herein several novel findings. First, that activated T lymphocytes from Graves' patients drive the differentiation of PPAR-gamma-expressing orbital fibroblasts to adipocytes. Second, this adipogenic differentiation is blocked by nonselective small molecule cyclooxygenase (Cox)-1/Cox-2 inhibitors and by Cox-2 selective inhibitors. Third, activated, but not naïve, human T cells highly express Cox-2 and synthesize prostaglandin D2 and related prostaglandins that are PPAR-gamma ligands. These provocative new findings provide evidence for how activated T lymphocytes, through production of PPAR-gamma ligands, profoundly influence human fibroblast differentiation to adipocytes. They also suggest the possibility that, in addition to the orbit, T lymphocytes influence the deposition of fat in other tissues.

The adipocyte as an important target cell for Trypanosoma cruzi infection.

Adipose tissue plays an active role in normal metabolic homeostasis as well as in the development of human disease. Beyond its obvious role as a depot for triglycerides, adipose tissue controls energy expenditure through secretion of several factors. Little attention has been given to the role of adipocytes in the pathogenesis of Chagas disease and the associated metabolic alterations. Our previous studies have indicated that hyperglycemia significantly increases parasitemia and mortality in mice infected with Trypanosoma cruzi. We determined the consequences of adipocyte infection in vitro and in vivo. Cultured 3T3-L1 adipocytes can be infected with high efficiency. Electron micrographs of infected cells revealed a large number of intracellular parasites that cluster around lipid droplets. Furthermore, infected adipocytes exhibited changes in expression levels of a number of different adipocyte-specific or adipocyte-enriched proteins. The adipocyte is therefore an important target cell during acute Chagas disease. Infection of adipocytes by T. cruzi profoundly influences the pattern of adipokines. During chronic infection, adipocytes may represent an important long-term reservoir for parasites from which relapse of infection can occur. We have demonstrated that acute infection has a unique metabolic profile with a high degree of local inflammation in adipose tissue, hypoadiponectinemia, hypoglycemia, and hypoinsulinemia but with relatively normal glucose disposal during an oral glucose tolerance test.