Shear stress-regulated miR-27b controls pericyte recruitment by repressing SEMA6A and SEMA6D.

AIMS: Vessel maturation involves the recruitment of mural cells such as pericytes and smooth muscle cells. Laminar shear stress is a major trigger for vessel maturation, but the molecular mechanisms by which shear stress affects recruitment of pericytes are unclear. MicroRNAs (miRs) are small non-coding RNAs, which post-transcriptionally control gene expression. The aim of the present study was to unveil the mechanism by which shear stress-regulated microRNAs contribute to vessel maturation. METHODS AND RESULTS: Here, we show that laminar shear stress increased miR-27a and miR-27b expression in vitro and in ex vivo in mouse femoral artery explants. Overexpression of miR-27b in endothelial cells increased pericyte adhesion and pericyte recruitment in vitro. In vitro barrier function of endothelial-pericyte co-cultures was augmented by miR-27b overexpression, whereas inhibition of miR-27a/b reduced adhesion and pericyte coverage and decreased barrier functions. In vivo, pharmacological inhibition of miR-27a/b by locked nucleic acid antisense oligonucleotides significantly reduced pericyte coverage and increased water content in the murine uterus. MiR-27b overexpression repressed semaphorins (SEMA), which mediate repulsive signals, and the vessel destabilizing human but not mouse Angiopoietin-2 (Ang-2). Silencing of SEMA6A and SEMA6D rescued the reduced pericyte adhesion by miR-27 inhibition. Furthermore, inhibition of SEMA6D increased barrier function of an endothelial-pericyte co-culture in vitro. CONCLUSION: The present study demonstrates for the first time that shear stress-regulated miR-27b promotes the interaction of endothelial cells with pericytes, partly by repressing SEMA6A and SEMA6D.

JMJD8 Regulates Angiogenic Sprouting and Cellular Metabolism by Interacting With Pyruvate Kinase M2 in Endothelial Cells.

OBJECTIVE: Jumonji C (JmjC) domain-containing proteins modify histone and nonhistone proteins thereby controlling cellular functions. However, the role of JmjC proteins in angiogenesis is largely unknown. Here, we characterize the expression of JmjC domain-containing proteins after inducing endothelial differentiation of murine embryonic stem cells and study the function of JmjC domain-only proteins in endothelial cell (EC) functions. APPROACH AND RESULTS: We identified a large number of JmjC domain-containing proteins regulated by endothelial differentiation of murine embryonic stem cells. Among the family of JmjC domain-only proteins, Jmjd8 was significantly upregulated on endothelial differentiation. Knockdown of Jmjd8 in ECs significantly decreased in vitro network formation and sprouting in the spheroid assay. JMJD8 is exclusively detectable in the cytoplasm, excluding a function as a histone-modifying enzyme. Mass spectrometry analysis revealed JMJD8-interacting proteins with known functions in cellular metabolism like pyruvate kinase M2. Accordingly, knockdown of pyruvate kinase M2 in human umbilical vein ECs decreased endothelial sprouting in the spheroid assay. Knockdown of JMJD8 caused a reduction of EC metabolism as measured by Seahorse Bioscience extracellular flux analysis. Conversely, overexpression of JMJD8 enhanced cellular oxygen consumption rate of ECs, reflecting an increased mitochondrial respiration. CONCLUSIONS: Jmjd8 is upregulated during endothelial differentiation and regulates endothelial sprouting and metabolism by interacting with pyruvate kinase M2.

MicroRNA-30 mediates anti-inflammatory effects of shear stress and KLF2 via repression of angiopoietin 2.

MicroRNAs are endogenously expressed small noncoding RNAs that regulate gene expression. Laminar blood flow induces atheroprotective gene expression in endothelial cells (ECs) in part by upregulating the transcription factor KLF2. Here, we identified KLF2- and flow-responsive miRs that affect gene expression in ECs. Bioinformatic assessment of mRNA expression patterns identified the miR-30-5p seed sequence to be highly enriched in mRNAs that are downregulated by KLF2. Indeed, KLF2 overexpression and shear stress stimulation in vitro and in vivo increased the expression of miR-30-5p family members. Furthermore, we identified angiopoietin 2 (Ang2) as a target of miR-30. MiR-30 overexpression reduces Ang2 levels, whereas miR-30 inhibition by LNA-antimiRs induces Ang2 expression. Consistently, miR-30 reduced basal and TNF-α-induced expression of the inflammatory cell­cell adhesion molecules E-selectin, ICAM1 and VCAM1, which was rescued by stimulation with exogenous Ang2. In summary, KLF2 and shear stress increase the expression of the miR-30-5p family which acts in an anti-inflammatory manner in ECs by impairing the expression of Ang2 and inflammatory cell­cell adhesion molecules. The upregulation of miR-30-5p family members may contribute to the atheroprotective effects of shear stress.

VEGF, Notch and TGFß/BMPs in regulation of sprouting angiogenesis and vascular patterning.

The blood vasculature is constantly adapting to meet the demand from tissue. In so doing, branches may form, reorganize or regress. These complex processes employ integration of multiple signalling cascades, some of them being restricted to endothelial and mural cells and, hence, suitable for targeting of the vasculature. Both genetic and drug targeting experiments have demonstrated the requirement for the vascular endothelial growth factor (VEGF) system, the Delta-like-Notch system and the transforming growth factor ß (TGFß)/bone morphogenetic protein (BMP) cascades in vascular development. Although several of these signalling cascades in part converge into common downstream components, they differ in temporal and spatial regulation and expression. For example, the pro-angiogenic VEGFA is secreted by cells in need of oxygen, presented to the basal side of the endothelium, whereas BMP9 and BMP10 are supplied via the bloodstream in constant interaction with the apical side to suppress angiogenesis. Delta-like 4 (DLL4), on the other hand, is provided as an endothelial membrane bound ligand. In the present article, we discuss recent data on the integration of these pathways in the process of sprouting angiogenesis and vascular patterning and malformation.

The hippocampal neurovascular niche during normal development and after irradiation to the juvenile mouse brain.

PURPOSE: To investigate the effects of cranial irradiation on the neurovascular niche in the young brain. Disruption of this niche has previously been observed in the adult rat brain after irradiation. MATERIALS AND METHODS: We subjected postnatal day 14 (P14) mice to a single dose of 8 Gy whole brain irradiation and measured the distance between microvessels and either neural progenitor cells (doublecortin-positive, DCX(+)) or proliferating cells (Ki-67(+)) in the dorsal hippocampal subgranular zone (SGZ) 6 hours, 1 week and 7 weeks post-irradiation. In addition, pericyte coverage of microvessels in the SGZ was measured. RESULTS: DCX(+) and Ki-67(+) cells were located closer to microvessels in the adult brain compared to young, still growing brains, constituting new information on normal development. We found an increased distance between microvessels and DCX(+) cells 6 h post-irradiation and between microvessels and Ki-67(+) cells 1 week post-irradiation. Furthermore, pericyte coverage was transiently decreased by 17% 6 h post-irradiation. CONCLUSIONS: The hippocampal neurovascular niche in the young, growing brain is transiently disrupted by irradiation. It remains to be elucidated what role these transient changes play in the apparently permanent ablation of hippocampal neurogenesis previously demonstrated in the same model.

MicroRNA-34a regulates cardiac ageing and function.

Ageing is the predominant risk factor for cardiovascular diseases and contributes to a significantly worse outcome in patients with acute myocardial infarction. MicroRNAs (miRNAs) have emerged as crucial regulators of cardiovascular function and some miRNAs have key roles in ageing. We propose that altered expression of miRNAs in the heart during ageing contributes to the age-dependent decline in cardiac function. Here we show that miR-34a is induced in the ageing heart and that in vivo silencing or genetic deletion of miR-34a reduces age-associated cardiomyocyte cell death. Moreover, miR-34a inhibition reduces cell death and fibrosis following acute myocardial infarction and improves recovery of myocardial function. Mechanistically, we identified PNUTS (also known as PPP1R10) as a novel direct miR-34a target, which reduces telomere shortening, DNA damage responses and cardiomyocyte apoptosis, and improves functional recovery after acute myocardial infarction. Together, these results identify age-induced expression of miR-34a and inhibition of its target PNUTS as a key mechanism that regulates cardiac contractile function during ageing and after acute myocardial infarction, by inducing DNA damage responses and telomere attrition.

The polarity protein Scrib is essential for directed endothelial cell migration.

RATIONALE: Polarity proteins are involved in the apico-basal orientation of epithelial cells, but relatively little is known regarding their function in mesenchymal cells. OBJECTIVE: We hypothesized that polarity proteins also contribute to endothelial processes like angiogenesis. METHODS AND RESULTS: Screening of endothelial cells revealed high expression of the polarity protein Scribble (Scrib). On fibronectin-coated carriers Scrib siRNA (siScrib) blocked directed but not random migration of human umbilical vein endothelial cells and led to an increased number and disturbed orientation of cellular lamellipodia. Coimmunoprecipitation/mass spectrometry and glutathione S-transferase (GST) pulldown assays identified integrin α5 as a novel Scrib interacting protein. By total internal reflection fluorescence (TIRF) microscopy, Scrib and integrin α5 colocalize at the basal plasma membrane of endothelial cells. Western blot and fluorescence activated cell sorting (FACS) analysis revealed that silencing of Scrib reduced the protein amount and surface expression of integrin α5 whereas surface expression of integrin αV was unaffected. Moreover, in contrast to fibronectin, the ligand of integrin α5, directional migration on collagen mediated by collagen-binding integrins was unaffected by siScrib. Mechanistically, Scrib supported integrin α5 recycling and protein stability by blocking its interaction with Rab7a, its translocation into lysosomes, and its subsequent degradation by pepstatin-sensitive proteases. In siScrib-treated cells, reinduction of the wild-type protein but not of PSD95, Dlg, ZO-1 (PDZ), or leucine rich repeat domain mutants restored integrin α5 abundance and directional cell migration. The downregulation of Scrib function in Tg(kdrl:EGFP)(s843) transgenic zebrafish embryos delayed the angiogenesis of intersegmental vessels. CONCLUSIONS: Scrib is a novel regulator of integrin α5 turnover and sorting, which is required for oriented cell migration and sprouting angiogenesis.

Histone deacetylase 9 promotes angiogenesis by targeting the antiangiogenic microRNA-17-92 cluster in endothelial cells.

OBJECTIVE: Histone deacetylases (HDACs) modulate gene expression by deacetylation of histone and nonhistone proteins. Several HDACs control angiogenesis, but the role of HDAC9 is unclear. METHODS AND RESULTS: Here, we analyzed the function of HDAC9 in angiogenesis and its involvement in regulating microRNAs. In vitro, silencing of HDAC9 reduces endothelial cell tube formation and sprouting. Furthermore, HDAC9 silencing decreases vessel formation in a spheroid-based Matrigel plug assay in mice and disturbs vascular patterning in zebrafish embryos. Genetic deletion of HDAC9 reduces retinal vessel outgrowth and impairs blood flow recovery after hindlimb ischemia. Consistently, overexpression of HDAC9 increases endothelial cell sprouting, whereas mutant constructs lacking the catalytic domain, the nuclear localization sequence, or sumoylation site show no effect. To determine the mechanism underlying the proangiogenic effect of HDAC9, we measured the expression of the microRNA (miR)-17-92 cluster, which is known for its antiangiogenic activity. We demonstrate that silencing of HDAC9 in endothelial cells increases the expression of miR-17-92. Inhibition of miR-17-20a rescues the sprouting defects induced by HDAC9 silencing in vitro and blocking miR-17 expression partially reverses the disturbed vascular patterning of HDAC9 knockdown in zebrafish embryos. CONCLUSIONS: We found that HDAC9 promotes angiogenesis and transcriptionally represses the miR-17-92 cluster.

MicroRNA-27a/b controls endothelial cell repulsion and angiogenesis by targeting semaphorin 6A.

MicroRNAs (miRs) are small RNAs that regulate gene expression at the posttranscriptional level. miR-27 is expressed in endothelial cells, but the specific functions of miR-27b and its family member miR-27a are largely unknown. Here we demonstrate that overexpression of miR-27a and miR-27b significantly increased endothelial cell sprouting. Inhibition of both miR-27a and miR-27b impaired endothelial cell sprout formation and induced endothelial cell repulsion in vitro. In vivo, inhibition of miR-27a/b decreased the number of perfused vessels in Matrigel plugs and impaired embryonic vessel formation in zebrafish. Mechanistically, miR-27 regulated the expression of the angiogenesis inhibitor semaphorin 6A (SEMA6A) in vitro and in vivo and targeted the 3'-untranslated region of SEMA6A. Silencing of SEMA6A partially reversed the inhibition of endothelial cell sprouting and abrogated the repulsion of endothelial cells mediated by miR-27a/b inhibition, indicating that SEMA6A is a functionally relevant miR-27 downstream target regulating endothelial cell repulsion. In summary, we show that miR-27a/b promotes angiogenesis by targeting the angiogenesis inhibitor SEMA6A, which controls repulsion of neighboring endothelial cells.

Class IIb HDAC6 regulates endothelial cell migration and angiogenesis by deacetylation of cortactin.

Histone deacetylases (HDACs) deacetylate histones and non-histone proteins, thereby affecting protein activity and gene expression. The regulation and function of the cytoplasmic class IIb HDAC6 in endothelial cells (ECs) is largely unexplored. Here, we demonstrate that HDAC6 is upregulated by hypoxia and is essential for angiogenesis. Silencing of HDAC6 in ECs decreases sprouting and migration in vitro and formation of functional vascular networks in matrigel plugs in vivo. HDAC6 regulates zebrafish vessel formation, and HDAC6-deficient mice showed a reduced formation of perfused vessels in matrigel plugs. Consistently, overexpression of wild-type HDAC6 increases sprouting from spheroids. HDAC6 function requires the catalytic activity but is independent of ubiquitin binding and deacetylation of α-tubulin. Instead, we found that HDAC6 interacts with and deacetylates the actin-remodelling protein cortactin in ECs, which is essential for zebrafish vessel formation and which mediates the angiogenic effect of HDAC6. In summary, we show that HDAC6 is necessary for angiogenesis in vivo and in vitro, involving the interaction and deacetylation of cortactin that regulates EC migration and sprouting.

HDAC5 is a repressor of angiogenesis and determines the angiogenic gene expression pattern of endothelial cells.

Class IIa histone deacetylases (HDACs) are signal-responsive regulators of gene expression involved in vascular homeostasis. To investigate the differential role of class IIa HDACs for the regulation of angiogenesis, we used siRNA to specifically suppress the individual HDAC isoenzymes. Silencing of HDAC5 exhibited a unique pro-angiogenic effect evidenced by increased endothelial cell migration, sprouting, and tube formation. Consistently, overexpression of HDAC5 decreased sprout formation, indicating that HDAC5 is a negative regulator of angiogenesis. The antiangiogenic activity of HDAC5 was independent of myocyte enhancer factor-2 binding and its deacetylase activity but required a nuclear localization indicating that HDAC5 might affect the transcriptional regulation of gene expression. To identify putative HDAC5 targets, we performed microarray expression analysis. Silencing of HDAC5 increased the expression of fibroblast growth factor 2 (FGF2) and angiogenic guidance factors, including Slit2. Antagonization of FGF2 or Slit2 reduced sprout induction in response to HDAC5 siRNA. Chromatin immunoprecipitation assays demonstrate that HDAC5 binds to the promoter of FGF2 and Slit2. In summary, HDAC5 represses angiogenic genes, such as FGF2 and Slit2, which causally contribute to capillary-like sprouting of endothelial cells. The derepression of angiogenic genes by HDAC5 inactivation may provide a useful therapeutic target for induction of angiogenesis.