MicroRNA-145 Regulates the Differentiation of Adipose Stem Cells Toward Microvascular Endothelial Cells and Promotes Angiogenesis.

RATIONALE: Adipose-derived stem cells (ASCs) are a potential adult mesenchymal stem cell source for restoring endothelial function in ischemic tissues. However, the mechanism that promotes ASCs differentiation toward endothelial cells (ECs) is not known. OBJECTIVE: To investigate the mechanisms of ASCs differentiation into ECs. METHODS AND RESULTS: ASCs were isolated from clinical lipoaspirates and cultured with DMEM or endothelial cell-conditioned medium. Endothelial cell-conditioned medium induced downregulation of miR-145 in ASCs and promoted endothelial differentiation. We identified bFGF (basic fibroblast growth factor) released by ECs as inducer of ASCs differentiation through receptor-induced AKT (protein kinase B) signaling and phosphorylation of FOXO1 (forkhead box protein O1) suppressing its transcriptional activity and decreasing miR-145 expression. Blocking bFGF-receptor or PI3K/AKT signaling in ASCs increased miR-145 levels. Modulation of miR-145 in ASCs, using a miR-145 inhibitor, regulated their differentiation into ECs: increasing proliferation, migration, inducing expression of EC markers (VE-cadherin, VEGFR2 [vascular endothelial growth factor receptor 2], or VWF [von Willebrand Factor]), and tube-like formation. Furthermore, in vivo, downregulation of miR-145 in ASCs enhanced angiogenesis in subcutaneously implanted plugs in mice. In a murine hindlimb ischemia model injection of ASCs with downregulated miR-145 induced collateral flow and capillary formation evidenced by magnetic resonance angiography. Next, we identified ETS1 (v-ets avian erythroblastosis virus E26 oncogene homolog 1) as the target of miR-145. Upregulation of miR-145 in ASCs, by mimic miR-145, suppressed ETS1 expression and consequently abolished EC differentiation and the angiogenic properties of endothelial cell-conditioned medium-preconditioned ASCs; whereas, overexpression of ETS1 reversed the abrogated antiangiogenic capacity of miR-145. ETS1 overexpression induced similar results to those obtained with miR-145 knockdown. CONCLUSIONS: bFGF released by ECs induces ASCs differentiation toward ECs through miR-145-regulated expression of ETS1. Downregulation of miR-145 in ASCs induce vascular network formation in ischemic muscle.

Macrophages of genetically characterized familial hypercholesterolaemia patients show up-regulation of LDL-receptor-related proteins.

Familial hypercholesterolaemia (FH) is a major risk for premature coronary heart disease due to severe long-life exposure to high LDL levels. Accumulation of LDL in the vascular wall triggers atherosclerosis with activation of the innate immunity system. Here, we have investigated (i) gene expression of LDLR and LRPs in peripheral blood cells (PBLs) and in differentiated macrophages of young FH-patients; and (ii) whether macrophage from FH patients have a differential response when exposed to high levels of atherogenic LDL. PBLs in young heterozygous genetically characterized FH patients have higher expression of LRP5 and LRP6 than age-matched healthy controls or patients with secondary hypercholesterolaemia. LRP1 levels were similar among groups. In monocyte-derived macrophages (MACs), LRP5 and LRP1 transcript levels did not differ between FHs and controls in resting conditions, but when exposed to agLDL, FH-MAC showed a highly significant up-regulation of LRP5, while LRP1 was unaffected. PBL and MAC cells from FH patients had significantly lower LDLR expression than control cells, independently of the lipid-lowering therapy. Furthermore, exposure of FH-MAC to agLDL resulted in a reduced expression of CD163, scavenger receptor with anti-inflammatory and atheroprotective properties. In summary, our results show for first time that LRPs, active lipid-internalizing receptors, are up-regulated in innate immunity cells of young FH patients that have functional LDLR mutations. Additionally, their reduced CD163 expression indicates less atheroprotection. Both mechanisms may play a synergic effect on the onset of premature atherosclerosis in FH patients.

Fibulin-5 is up-regulated by hypoxia in endothelial cells through a hypoxia-inducible factor-1 (HIF-1α)-dependent mechanism.

Hypoxia modulates gene expression and affects multiple aspects of endothelial cell biology. Fibulin-5 (FBLN5) is an extracellular matrix protein essential for elastic fiber assembly and vasculogenesis that participates in vascular remodeling and controls endothelial cell adhesion, motility, and proliferation. In this context, we aimed to analyze FBLN5 regulation by hypoxia in endothelial cells. Hypoxia (1% O(2)) increased FBLN5 mRNA levels in endothelial cells in a time-dependent manner. Maximal induction (∼2.5-fold) was achieved after 24 h of hypoxia. This effect paralleled an increase in both intracellular and extracellular FBLN5 protein levels. The increase in FBLN5 mRNA levels observed in hypoxic cells was blocked by inhibitors of the PI3K/Akt/mTOR pathway (LY294002 and rapamycin) and mimicked by dimethyl oxal glycine, which prevents proline hydroxylase-mediated degradation of HIF-1α. Silencing of HIF-1α completely prevented hypoxia-induced FBLN5 up-regulation. Accordingly, both hypoxia and HIF-1α overexpression increased FBLN5 transcriptional activity. Serial promoter deletion and mutagenesis studies revealed the involvement of a putative hypoxia response element (HRE) located at -78 bp. In fact, EMSA and ChIP assays demonstrated increased HIF-1 binding to this site in hypoxic cells. Interestingly, the rate of endothelial cells undergoing apoptosis in cultures exposed to hypoxia increased in FBLN5 knockdown cells, suggesting that hypoxia-induced FBLN5 expression contributes to preserve cell survival. These results provide evidence that HIF-1 signaling underlies the increase of FBLN5 expression elicited by hypoxia in endothelial cells and suggest that FBLN5 induction could be involved in the adaptive survival response of endothelial cells to hypoxia.

Ets-1 transcription is required in tissue factor driven microvessel formation and stabilization.

Tissue factor (TF) has well-recognized roles as initiator of blood coagulation as well as an intracellular signaling receptor. TF signaling regulates gene transcription and protein translation. Recently, we have shown that TF-induced mature neovessel formation is ultimately driven by CCL2 expression. However, the signaling process induced by TF to promote microvessel formation remains to be determined. This study was designed with the objective to investigate the mechanisms involved in TF-induced neovessel formation. Here, we have identified that Ets-1 expression is a downstream effector of TF signaling. TF-siRNA induced a highly significant reduction in Ets-1 expression levels and in Ets-1/DNA binding while inducing abrogation of microvessel formation. Activation of Ets-1 rescued the effect of TF inhibition and restored microvessel formation confirming the critical role of Ets-1 in TF-induced angiogenesis. VE-cadherin expression, a key regulator of endothelial intercellular junctions, and an Ets-1 target molecule was dependent of TF-inhibition. We show that TF signals through ERK1/2 to activate Ets-1 and induce CCL2 gene expression by binding to its promoter region. We conclude that endothelial cell TF signals through ERK1/2 and Ets-1 to trigger microvessel formation.

Tissue factor regulates microvessel formation and stabilization by induction of chemokine (C-C motif) ligand 2 expression.

OBJECTIVE: Tissue factor (TF) triggers arterial thrombosis. TF is also able to initiate cellular signaling mechanisms leading to angiogenesis. Because high cardiovascular risk atherosclerotic plaques show significant angiogenesis, our objective was to investigate whether TF is able to trigger and stabilize atherosclerotic plaque neovessel formation. METHODS AND RESULTS: In this study, we showed, by real-time confocal microscopy in 3-dimensional basement membrane cocultures, that TF in human microvascular endothelial cells (HMEC-1) and in human vascular smooth muscle cells (HVSMCs) plays an important role in the formation of capillary-like networks. TF silencing in endothelial cells and smooth muscle cells inhibits the formation of tube-like structures with stable phenotype. Using an in vivo model, we observed that TF inhibition in either HMEC-1 or HVSMCs reduced their shared ability to form new capillaries. The phenotypic changes induced by TF silencing were linked to reduced chemokine (C-C motif) ligand 2 (CCL2) expression in endothelial cells. Wound healing and chemotactic assays demonstrated that TF-induced release of CCL2 stimulated HVSMC migration to HMEC-1. CONCLUSION: Endogenous TF regulates CCL2 production in endothelial cells. Secreted CCL2 mediates the angiogenic effect of TF by recruiting smooth muscle cells toward endothelial cells and facilitates the maturation of newly formed microvessels.

Hypoxia stimulates low-density lipoprotein receptor-related protein-1 expression through hypoxia-inducible factor-1α in human vascular smooth muscle cells.

OBJECTIVE: Hypoxia is considered a key factor in the progression of atherosclerotic lesions. Low-density lipoprotein receptor-related protein (LRP1) plays a pivotal role in the vasculature. The aim of this study was to investigate the effect of hypoxia on LRP1 expression and function in vascular smooth muscle cells (VSMC) and the role of hypoxia-inducible factor-α (HIF-1α). METHODS AND RESULTS: Real-time polymerase chain reaction and Western blot analysis demonstrated that hypoxia (1% O(2)) time-dependently induced LRP1 mRNA (maximum levels at 1 to 2 hours) and protein expression (maximum levels at 12 to 24 hours). The delayed hypoxic upregulation of LRP1 protein versus mRNA may be explained by the long half-life of LRP1 protein. Luciferase assays demonstrated that hypoxia and HIF-1α overaccumulation induced LRP1 promoter activity and that 2 consensus hypoxia response element sites located at -1072/-1069 and -695/-692 participate in the induction. Chromatin immunoprecipitation showed the in vivo binding of HIF-1α to LRP1 promoter in hypoxic VSMC. Hypoxia effects on LRP1 protein expression were functionally translated into an increased cholesteryl ester (CE) accumulation from aggregated low-density lipoprotein (agLDL) uptake. The blockade of HIF-1α expression inhibited the upregulatory effect of hypoxia on LRP1 expression and agLDL-derived intracellular CE overaccumulation, suggesting that both LRP1 overexpression and CE overaccumulation in hypoxic vascular cells are dependent on HIF-1α. Immunohistochemical analysis showed the colocalization of LRP1 and HIF-1α in vascular cells of human advanced atherosclerotic plaques. CONCLUSION: Hypoxia upregulates LRP1 expression and agLDL-derived intracellular CE accumulation in human VSMC through HIF-1α induction.

PAR2-SMAD3 in microvascular endothelial cells is indispensable for vascular stability via tissue factor signaling.

Tissue factor (TF) signaling regulates gene expression and protein synthesis leading to the modulation of cell function. Recently, we have demonstrated in microvascular endothelial cells (mECs) that TF signaling induces activation of ETS1 transcription factor. Because combinatorial control is a characteristic property of ETS family members, involving the interaction between ETS1 and other transcription factors, here we investigate whether additional transcription factors are involved in TF-induced angiogenesis. We show by in vitro and in vivo experiments that in addition to ETS1, SMAD3 contributes to tube-like stabilization induced by TF in mECs. Whereas the ability of TF-overexpressing cells to induce gene expression through ETS1 is dependent on AKT signaling, SMAD3 induces ETS1 by an alternative AKT-independent pathway. Moreover, while TF-AKT-ETS1 pathway to induce CCL2 is PAR2-independent, PAR2 is required for TF-SMAD3-induced CCL2 expression. PAR2-dependent activation of SMAD3 is mediated by PKC phosphorylation. In addition, disruption of SMAD3 expression in mECs reduces ERK1/2 phosphorylation and decreases target gene promoter activity. In conclusion, in mECs TF-induced angiogenesis seems to be the result of two signaling pathways: TF-induced microvessel formation is regulated through ß1 integrin-AKT-ETS1; and TF-induced microvessel stabilization is regulated via PAR2-SMAD3 that is indispensable for the maintenance of vascular integrity.

rs11613352 polymorphism (TT genotype) associates with a decrease of triglycerides and an increase of HDL in familial hypercholesterolemia patients.

INTRODUCTION AND OBJECTIVES: Recent genome-wide association studies have identified a locus on chromosome 12q13.3 associated with plasma levels of triglyceride and high-density lipoprotein cholesterol, with rs11613352 being the lead single nucleotide polymorphism in this genome-wide association study locus. The aim of the study is to investigate the involvement of rs11613352 in a population with high cardiovascular risk due to familial hypercholesterolemia. METHODS: The single nucleotide polymorphism was genotyped by Taqman(®) assay in a cohort of 601 unrelated familial hypercholesterolemia patients and its association with plasma triglyceride and high-density lipoprotein cholesterol levels was analyzed by multivariate methods based on linear regression. RESULTS: Minimal allele frequency was 0.17 and genotype frequencies were 0.69, 0.27, and 0.04 for CC, CT, and TT genotypes, respectively. The polymorphism is associated in a recessive manner (TT genotype) with a decrease in triglyceride levels (P=.002) and with an increase in high-density lipoprotein cholesterol levels (P=.021) after adjusting by age and sex. CONCLUSIONS: The polymorphism rs11613352 may contribute to modulate the cardiovascular risk by modifying plasma lipid levels in familial hypercholesterolemia patients.

CDP-choline prevents glutamate-mediated cell death in cerebellar granule neurons.

Cytidine 5'-diphosphocholine (CDP-choline) has been shown to reduce neuronal degeneration induced in central nervous system (CNS) injury. However, the precise mechanism underlying the neuroprotective properties of this molecule is still unknown. Excitotoxicity causes cell death in CNS injury (trauma or ischemia) and has also been involved in neurodegenerative diseases. We have examined whether CDP-choline prevents glutamate-mediated cell death, determined by trypan blue exclusion and lactate dehydrogenase activity assays. Pretreatment of rat cerebellar granule cells (CGCs) with CDP-choline causes a dose- and time-dependent reduction of glutamate-induced excitotoxicity. Cell death is prevented >50% when 100 microM CDP-choline is added 6 d before the glutamate excitotoxic insult but less than 20% when added concomitantly with glutamate. Pretreatment of CGCs with CDP-choline reduces almost completely (>80%) the number of apoptotic cells analyzed by flow cytometry, suggesting that CDP-choline exerts a neuroprotective effect by inhibiting the apoptotic pathway induced by glutamate.

Monocyte-secreted Wnt5a interacts with FZD5 in microvascular endothelial cells and induces angiogenesis through tissue factor signaling.

Angiogenesis during reactive and pathologic processes is characteristically associated with inflammation. Inflammatory cells participate in angiogenesis by secreting different molecules that affect endothelial cell functions. We had previously shown that induced tissue factor (TF) expression in activated microvascular endothelial cells (mEC) is able to induce angiogenesis via autocrine regulation. However, the signals that induce TF expression in mEC are not fully known. Here, we demonstrate that monocyte paracrine cross-talk with mECs triggers mEC-TF expression. We have identified that monocyte-secreted Wnt5a induces TF expression in mEC and functionally induces cell monolayer repair and angiotube formation in vitro as well as microvessel formation in vivo. Monocyte-secreted Wnt5a activates FZD5 in mECs, which signals to induce the release of intracellular Ca(2+) and increase NFκB transcription activity and TF gene expression. In sum, Wnt5a secreted by monocytes signals through the noncanonical Wnt-FZD5 pathway in mECs to induce TF expression that induces angiogenesis by autocrine regulation.

LRP1 gene polymorphisms are associated with premature risk of cardiovascular disease in patients with familial hypercholesterolemia.

INTRODUCTION AND OBJECTIVES: LRP1 gene overexpression in atherosclerotic plaque is associated with increased lipid uptake through the vascular wall. The aim of the study was to analyze whether LRP1 modulates the genetic risk of developing premature cardiovascular disease in familial hypercholesterolemia, using single nucleotide polymorphism association analysis. METHODS: Ten polymorphisms of the LRP1 gene (rs715948, rs1799986, rs1800127, rs7968719, rs1800176, rs1800194, rs1800181, rs1140648, rs1800164, and rs35282763) were genotyped in 339 patients (77 with premature cardiovascular disease and 262 without) in the SAFEHEART study. RESULTS: The c.677C>T (rs1799986) polymorphism showed a significant association with premature cardiovascular disease after adjusting by sex, age, body mass index, and the effect of the low-density lipoprotein receptor mutation in the dominant model (CT+TT vs CC: odds ratio=1.94; 95% confidence interval, 1.08-3.48; P=.029). Similar results were observed after increasing the sample to 648 subjects (133 with premature cardiovascular disease vs 515 without [odds ratio=1.83; 95% confidence interval, 1.16-2.88; P=.011]). CONCLUSIONS: The c.677C>T polymorphism is associated with increased rates of premature cardiovascular disease in familial hypercholesterolemia. Although we were unable to show that this polymorphism was involved in the alteration of normal mRNA splicing patterns, the possibility that it is in strong linkage disequilibrium with another functional polymorphism cannot be ruled out and would explain the cause-effect relationship with cardiovascular disease risk in this population. Further studies are needed to replicate the results and to localize the putative genetic variants associated with this polymorphism.

Low-density lipoprotein receptor-related protein 1 mediates hypoxia-induced very low density lipoprotein-cholesteryl ester uptake and accumulation in cardiomyocytes.

AIMS: The myocardium accumulates intracellular lipids under ischaemic conditions, and myocardial fat deposition is closely associated with cardiac dysfunction. Our aims were to analyse the effect of hypoxia on low-density lipoprotein receptor-related protein 1 (LRP1) expression in neonatal rat ventricular myocytes (NRVM) and cardiac-derived HL-1 cells and the molecular mechanisms involved in this effect, to determine the role of LRP1 in the very low density lipoprotein (VLDL) uptake by hypoxic cardiomyocytes, and to study the effect of hypoxia on lipoprotein receptor expression and myocardial lipid profile in an in vivo porcine experimental model of acute myocardial infarction. METHODS AND RESULTS: Thin-layer chromatography after lipid extraction showed that VLDL exposure leads to cholesteryl ester (CE) and triglyceride (TG) accumulation in a dose-dependent manner and that hypoxic conditions further increased VLDL-derived intracellular lipid accumulation in HL-1 cells. Knockdown of LRP1 through lentiviral-mediated interfering RNA specifically prevented hypoxia-induced VLDL-CE internalization in HL-1 cells and NRVM. Lipopolysaccharide (LPS)-induced LRP1 overexpression specifically increased VLDL-CE accumulation in NRVM. In addition, using double-radiolabelled [(3)H]CE-[(14)C]TG-VLDL, we found that LRP1 deficiency specifically prevented hypoxia-induced VLDL-[(3)H]CE uptake. Finally, in an in vivo porcine model of infarcted myocardium, ischaemic areas exhibited LRP1 protein up-regulation and intramyocardial CE overaccumulation. CONCLUSION: Our results demonstrate that hypoxia increases LRP1 expression through HIF-1α and that LRP1 overexpression mediates hypoxia-induced VLDL-CE uptake and accumulation in cardiomyocytes.

The diagnosis of mitochondrial HMG-CoA synthase deficiency.

Deficiency of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase, the only disorder exclusively affecting hepatic ketogenesis, is a cause of hypoglycemic coma. We report that the diagnosis can be made by typical laboratory findings (hypoketosis, elevated free fatty acids, normal acylcarnitines, specific urinary organic acids) during acute episodes.

Structural (betaalpha)8 TIM barrel model of 3-hydroxy-3-methylglutaryl-coenzyme A lyase.

This study describes three novel homozygous missense mutations (S75R, S201Y, and D204N) in the 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase gene, which caused 3-hydroxy-3-methylglutaric aciduria in patients from Germany, England, and Argentina. Expression studies in Escherichia coli show that S75R and S201Y substitutions completely abolished the HMG-CoA lyase activity, whereas D204N reduced catalytic efficiency to 6.6% of the wild type. We also propose a three-dimensional model for human HMG-CoA lyase containing a (betaalpha)8 (TIM) barrel structure. The model is supported by the similarity with analogous TIM barrel structures of functionally related proteins, by the localization of catalytic amino acids at the active site, and by the coincidence between the shape of the substrate (HMG-CoA) and the predicted inner cavity. The three novel mutations explain the lack of HMG-CoA lyase activity on the basis of the proposed structure: in S75R and S201Y because the new amino acid residues occlude the substrate cavity, and in D204N because the mutation alters the electrochemical environment of the active site. We also report the localization of all missense mutations reported to date and show that these mutations are located in the beta-sheets around the substrate cavity.