BReast CAncer susceptibility gene 2 deficiency exacerbates oxidized LDL-induced DNA damage and endothelial apoptosis.

Mutations in the tumor suppressor gene BRCA2 (BReast CAncer susceptibility gene 2) predispose carriers to breast, ovarian, and other cancers. In response to DNA damage, BRCA2 participates in homology-directed DNA damage repair to maintain genome stability. Genome-wide association studies have identified an association between BRCA2 single nucleotide polymorphisms and plasma-lipid levels and lipid deregulation in humans. To date, DNA damage, apoptosis, and lipid deregulation are recognized as central pathways for endothelial dysfunction and atherosclerosis; however, the role of BRCA2 in endothelial dysfunction remains to be elucidated. To determine the role of BRCA2 in endothelial dysfunction, BRCA2 was silenced in human umbilical vein endothelial cells (ECs) and assessed for markers of DNA damage, apoptosis, and endothelial function following oxidized low-density lipoprotein (oxLDL) treatment. OxLDL was found to induce significant reactive oxygen species (ROS) production in BRCA2-silenced ECs. This increase in ROS production was associated with exacerbated DNA damage evidenced by increased expression and activation of DNA double-stranded break (DSB) marker γH2AX and reduced RAD51-foci formation-an essential regulator of DSB repair. Increased DSBs were associated with enhanced expression and activation of pro-apoptotic p53 and significant apoptosis in oxLDL-treated BRCA2-silenced ECs. Loss of BRCA2 in ECs was further associated with oxLDL-induced impaired tube-forming potential and eNOS expression. Collectively, the data reveals, for the first time, a novel role of BRCA2 as a regulator of EC survival and function in the setting of oxLDL treatment in vitro. Additionally, the data provide important clues regarding the potential susceptibility of BRCA2 mutation carriers to endothelial dysfunction, atherosclerosis, and other cardiovascular diseases.

Endothelial-specific Loss of IFT88 Promotes Endothelial-to-Mesenchymal Transition and Exacerbates Bleomycin-induced Pulmonary Fibrosis.

Intraflagellar transport protein 88 (Ift88) is required for ciliogenesis and shear stress-induced dissolution of cilia in embryonic endothelial cells coincides with endothelial-to-mesenchymal transition (EndMT) in the developing heart. EndMT is also suggested to underlie heart and lung fibrosis, however, the mechanism linking endothelial Ift88, its effect on EndMT and organ fibrosis remains mainly unexplored. We silenced Ift88 in endothelial cells (ECs) in vitro and generated endothelial cell-specific Ift88-knockout mice (Ift88endo) in vivo to evaluate EndMT and its contribution towards organ fibrosis, respectively. Ift88-silencing in ECs led to mesenchymal cells-like changes in endothelial cells. The expression level of the endothelial markers (CD31, Tie-2 and VE-cadherin) were significantly reduced with a concomitant increase in the expression level of mesenchymal markers (αSMA, N-Cadherin and FSP-1) in Ift88-silenced ECs. Increased EndMT was associated with increased expression of profibrotic Collagen I expression and increased proliferation in Ift88-silenced ECs. Loss of Ift88 in ECs was further associated with increased expression of Sonic Hedgehog signaling effectors. In vivo, endothelial cells isolated from the heart and lung of Ift88endo mice demonstrated loss of Ift88 expression in the endothelium. The Ift88endo mice were born in expected Mendelian ratios without any adverse cardiac phenotypes at baseline. Cardiac and pulmonary endothelial cells isolated from the Ift88endo mice demonstrated signs of EndMT and bleomycin treatment exacerbated pulmonary fibrosis in Ift88endo mice. Pressure overload stress in the form of aortic banding did not reveal a significant difference in cardiac fibrosis between Ift88endo mice and control mice. Our findings demonstrate a novel association between endothelial cilia with EndMT and cell proliferation and also show that loss of endothelial cilia-associated increase in EndMT contributes specifically towards pulmonary fibrosis.

Jack of many trades: Multifaceted role of neuropilins in pancreatic cancer.

Neuropilins (NRPs) have been described as receptors for class 3 semaphorins and coreceptors for a plethora of ligands, such as members of the vascular endothelial growth factor (VEGF) family of angiogenic cytokines and transforming growth factor (TGF). Initial studies using genetic models have indicated that neuropilin-1 (NRP-1) is essential for axonal guidance during neuronal and cardiovascular development, regulated via semaphorins and VEGF, respectively, whereas the other homolog of neuropilin, NRP-2, has been shown to play a more specific role in neuronal patterning and lymphangiogenesis. Pancreatic ductal adenocarcinoma (PDAC) remains a significant cause of cancer mortality with the lowest five-year survival rate compared to other types of cancer. Recent findings have indicated that NRPs are abundantly expressed in pancreatic cancer cell lines and pancreatic tumor tissues, where they mediate several essential cancer-initiating and cancer-promoting functional responses through their unique ability to bind multiple ligands. Specifically, NRPs have been implicated in numerous biological processes such as cancer cell proliferation, survival, invasion, and tumor growth. More recently, several other protumorigenic roles mediated by NRPs have emerged, advocating NRPs as ideal therapeutic targets against PDAC.

Valproic Acid Induces Endothelial-to-Mesenchymal Transition-Like Phenotypic Switching.

Valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, is a widely used anticonvulsant drug that is currently undergoing clinical evaluation for anticancer therapy due to its anti-angiogenic potential. Endothelial cells (ECs) can transition into mesenchymal cells and this form of EC plasticity is called endothelial-to-mesenchymal transition (EndMT), which is widely implicated in several pathologies including cancer and organ fibrosis. However, the effect of VPA on EC plasticity and EndMT remains completely unknown. We report herein that VPA-treatment significantly inhibits tube formation, migration, nitric oxide production, proliferation and migration in ECs. A microscopic evaluation revealed, and qPCR, immunofluorescence and immunoblotting data confirmed EndMT-like phenotypic switching as well as an increased expression of pro-fibrotic genes in VPA-treated ECs. Furthermore, our data confirmed important and regulatory role played by TGFß-signaling in VPA-induced EndMT. Our qPCR array data performed for 84 endothelial genes further supported our findings and demonstrated 28 significantly and differentially regulated genes mainly implicated in angiogenesis, endothelial function, EndMT and fibrosis. We, for the first time report that VPA-treatment associated EndMT contributes to the VPA-associated loss of endothelial function. Our data also suggest that VPA based therapeutics may exacerbate endothelial dysfunction and EndMT-related phenotype in patients undergoing anticonvulsant or anticancer therapy, warranting further investigation.

Friends Turned Foes: Angiogenic Growth Factors beyond Angiogenesis.

Angiogenesis, the formation of new blood vessels from pre-existing ones is a biological process that ensures an adequate blood flow is maintained to provide the cells with a sufficient supply of nutrients and oxygen within the body. Numerous soluble growth factors and inhibitors, cytokines, proteases as well as extracellular matrix proteins and adhesion molecules stringently regulate the multi-factorial process of angiogenesis. The properties and interactions of key angiogenic molecules such as vascular endothelial growth factors (VEGFs), fibroblast growth factors (FGFs) and angiopoietins have been investigated in great detail with respect to their molecular impact on angiogenesis. Since the discovery of angiogenic growth factors, much research has been focused on their biological actions and their potential use as therapeutic targets for angiogenic or anti-angiogenic strategies in a context-dependent manner depending on the pathologies. It is generally accepted that these factors play an indispensable role in angiogenesis. However, it is becoming increasingly evident that this is not their only role and it is likely that the angiogenic factors have important functions in a wider range of biological and pathological processes. The additional roles played by these molecules in numerous pathologies and biological processes beyond angiogenesis are discussed in this review.

Endothelial long non-coding RNAs regulated by oxidized LDL.

Oxidized low-density lipoprotein (oxLDL) plays a central role in the pathogenesis of atherosclerosis, in part via an effect to promote endothelial dysfunction. In the present study, we evaluated the expression profiles of long non-coding RNAs (lncRNAs) and protein-coding mRNAs in endothelial cells following oxLDL stimulation. LncRNAs and mRNAs from human umbilical vein endothelial cells (HUVECs) were profiled with the Arraystar Human lncRNA Expression Microarray V3.0 following 24 h of oxLDL treatment (100 µg/mL). Of the 30,584 lncRNAs screened, 923 were significantly up-regulated and 975 significantly down-regulated (P < 0.05) in response to oxLDL exposure. In the same HUVEC samples, 518 of the 26,106 mRNAs screened were up-regulated and 572 were down-regulated. Of these differentially expressed lncRNAs, CLDN10-AS1 and CTC-459I6.1 were the most up-regulated (~87-fold) and down-regulated (~28-fold), respectively. Bioinformatic assignment of the differentially regulated genes into functional groups indicated that many are involved in signaling pathways among which are the cytokine receptor, chemokine, TNF, MAPK and Ras signaling pathways, olfactory transduction, and vascular smooth muscle cell function. This is the first report profiling oxLDL-mediated changes in lncRNA and mRNA expression in human endothelial cells. The novel targets revealed substantially extend the list of potential candidate genes involved in atherogenesis.

Cardiac Overexpression of S100A6 Attenuates Cardiomyocyte Apoptosis and Reduces Infarct Size After Myocardial Ischemia-Reperfusion.

BACKGROUND: Cardiomyocyte-specific transgenic mice overexpressing S100A6, a member of the family of EF-hand calcium-binding proteins, develop less cardiac hypertrophy, interstitial fibrosis, and myocyte apoptosis after permanent coronary ligation, findings that support S100A6 as a potential therapeutic target after acute myocardial infarction. Our purpose was to investigate S100A6 gene therapy for acute myocardial ischemia-reperfusion. METHODS AND RESULTS: We first performed in vitro studies to examine the effects of S100A6 overexpression and knockdown in rat neonatal cardiomyocytes. S100A6 overexpression improved calcium transients and protected against apoptosis induced by hypoxia-reoxygenation via enhanced calcineurin activity, whereas knockdown of S100A6 had detrimental effects. For in vivo studies, human S100A6 plasmid or empty plasmid was delivered to the left ventricular myocardium by ultrasound-targeted microbubble destruction in Fischer-344 rats 2 days prior to a 30-minute ligation of the left anterior descending coronary artery followed by reperfusion. Control animals received no therapy. Pretreatment with S100A6 gene therapy yielded a survival advantage compared to empty-plasmid and nontreated controls. S100A6-pretreated animals had reduced infarct size and improved left ventricular systolic function, with less myocyte apoptosis, attenuated cardiac hypertrophy, and less cardiac fibrosis. CONCLUSIONS: S100A6 overexpression by ultrasound-targeted microbubble destruction helps ameliorate myocardial ischemia-reperfusion, resulting in lower mortality and improved left ventricular systolic function post-ischemia-reperfusion via attenuation of apoptosis, reduction in cardiac hypertrophy, and reduced infarct size. Our results indicate that S100A6 is a potential therapeutic target for acute myocardial infarction.

Novel regulatory role of neuropilin-1 in endothelial-to-mesenchymal transition and fibrosis in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an intense fibrotic reaction termed tumor desmoplasia, which is in part responsible for its aggressiveness. Endothelial cells have been shown to display cellular plasticity in the form of endothelial-to-mesenchymal transition (EndMT) that serves as an important source of fibroblasts in pathological disorders, including cancer. Angiogenic co-receptor, neuropilin-1 (NRP- 1) actively binds TGFß1, the primary mediator of EndMT and is involved in oncogenic processes like epithelial-to-mesenchymal transition (EMT). NRP-1 and TGFß1 signaling have been shown to be aberrantly up-regulated in PDAC. We report herein a positive correlation between NRP-1 levels, EndMT and fibrosis in human PDAC xenografts. Loss of NRP-1 in HUVECs limited TGFß1-induced EndMT as demonstrated by gain of endothelial and loss of mesenchymal markers, while maintaining endothelial cell architecture. Knockdown of NRP-1 down-regulated TGFß canonical signaling (pSMAD2) and associated pro-fibrotic genes. Overexpression of NRP-1 exacerbated TGFß1-induced EndMT and up-regulated TGFß signaling and expression of pro-fibrotic genes. In vivo, loss of NRP-1 attenuated tumor perfusion and size, accompanied by reduction in EndMT and fibrosis. This study defines a previously unrecognized role of NRP-1 in regulating TGFß1-induced EndMT and fibrosis, and advocates NRP-1 as a therapeutic target to reduce tumor fibrosis and PDAC progression.

Investigation of novel LPS-induced differentially expressed long non-coding RNAs in endothelial cells.

The molecular mechanisms responsible for sepsis-induced endothelial dysfunction leading to an elevated risk of cardiovascular diseases remain undefined. Endotoxic or septic shock is a potentially lethal complication of systemic infection by Gram-negative bacteria. Lipopolysaccharide (LPS) is a critical glycolipid component of the outer wall of Gram-negative bacteria, and many of the sepsis-associated cellular signals by Gram-negative bacteria are attributed to LPS. Given that LPS has an established role in the pathophysiology of sepsis and long non-coding RNAs (lncRNAs) have been reported to critically regulate vascular homeostasis, a systematic transcriptional survey was conducted to evaluate the impact of LPS stimulation on human endothelial lncRNAs and protein-coding transcripts (mRNAs). LncRNAs and mRNAs from LPS-treated (100 ng/mL; 24 h) human umbilical vein endothelial cells (HUVECs) were profiled with the Arraystar Human lncRNA Expression Microarray V3.0. Of the 30,584 lncRNAs screened, 871 were significantly upregulated and 1068 significantly downregulated (p < 0.05) in response to LPS. In the same HUVEC samples, 733 of the 26,106 mRNAs screened were upregulated and 536 were downregulated. Among the differentially expressed lncRNAs, AL132709.5 was the most upregulated (~70 fold) and CTC-459I6.1 the most downregulated (~28 fold). Bioinformatics analyses indicated that the differentially expressed upregulated mRNAs are primarily enriched in cytokine-cytokine receptor interaction, infectious diseases, TNF signaling pathway, FoxO signaling pathway, and pathways in cancer. This is the first lncRNA and mRNA transcriptome profile of LPS-mediated changes in human endothelial cells. These observations may reveal novel endothelial targets of LPS that may be involved in the vascular pathology of sepsis.

Investigation of TGFß1-Induced Long Noncoding RNAs in Endothelial Cells.

Objective. To evaluate the relationship between TGFß signaling and endothelial lncRNA expression. Methods. Human umbilical vein endothelial cell (HUVECs) lncRNAs and mRNAs were profiled with the Arraystar Human lncRNA Expression Microarray V3.0 after 24 hours of exposure to TGFß1 (10 ng/mL). Results. Of the 30,584 lncRNAs screened, 2,051 were significantly upregulated and 2,393 were appreciably downregulated (P < 0.05) in response to TGFß. In the same HUVEC samples, 2,148 of the 26,106 mRNAs screened were upregulated and 1,290 were downregulated. Of these 2,051 differentially expressed upregulated lncRNAs, MALAT1, which is known to be induced by TGFß in endothelial cells, was the most (~220-fold) upregulated lncRNA. Bioinformatics analyses indicated that the differentially expressed upregulated mRNAs are primarily enriched in hippo signaling, Wnt signaling, focal adhesion, neuroactive ligand-receptor interaction, and pathways in cancer. The most downregulated are notably involved in olfactory transduction, PI3-Akt signaling, Ras signaling, neuroactive ligand-receptor interaction, and apoptosis. Conclusions. This is the first lncRNA and mRNA transcriptome profile of TGFß-mediated changes in human endothelial cells. These observations may reveal potential new targets of TGFß in endothelial cells and novel therapeutic avenues for cardiovascular disease-associated endothelial dysfunction.

Prospect of ultrasound-mediated gene delivery in cardiovascular applications.

INTRODUCTION: The field of regenerative medicine has evolved over the years, investigating gene and stem/progenitor cell therapies to help address the increasing burden of cardiovascular disease (CVD). While the lack of success of gene therapy in clinical trials has dampened enthusiasm, the search continues for a successful and translatable gene therapy strategy for CVD. Ultrasound-mediated gene delivery (UMGD) is a non-invasive technique for gene delivery that utilizes gene-bearing carrier microbubbles and high power ultrasound to facilitate transfection in vivo. Many pre-clinical studies have shown benefit in animal models of CVD, but this has yet to be translated to human applications. AREAS COVERED: In this review, the basic principles of UMGD will be examined along with an overview of pre-clinical studies to date in CVD, focusing on cardiac and vascular applications and key findings. In addition, the potential path to the clinical translation of UMGD is discussed. EXPERT OPINION: Ultrasound-mediated gene delivery holds promise as a non-invasive technique for gene delivery in CVD, with the ability to deliver multiple genes with repeated deliveries over time. If the substantial hurdles to clinical translation can be overcome, UMGD may prove to be a key aspect in the success of cardiovascular gene therapy in the future.

Microbubbles and Ultrasound: Therapeutic Applications in Diabetic Nephropathy.

Diabetic nephropathy (DN) remains one of the most common causes of end-stage renal disease. Current therapeutic strategies aiming at optimization of serum glucose and blood pressure are beneficial in early stage DN, but are unable to fully prevent disease progression. With the limitations of current medical therapies and the shortage of available donor organs for kidney transplantation, the need for novel therapies to address DN complications and prevent progression towards end-stage renal failure is crucial. The development of ultrasound technology for non-invasive and targeted in-vivo gene delivery using high power ultrasound and carrier microbubbles offers great therapeutic potential for the prevention and treatment of DN. The promising results from preclinical studies of ultrasound-mediated gene delivery (UMGD) in several DN animal models suggest that UMGD offers a unique, non-invasive platform for gene- and cell-based therapies targeted against DN with strong clinical translation potential.

Therapeutic Angiogenesis by Ultrasound-Mediated MicroRNA-126-3p Delivery.

OBJECTIVE: MicroRNAs are involved in many critical functions, including angiogenesis. Ultrasound-targeted microbubble destruction (UTMD) is a noninvasive technique for targeted vascular transfection of plasmid DNA and may be well suited for proangiogenic microRNA delivery. We aimed to investigate UTMD of miR-126-3p for therapeutic angiogenesis in chronic ischemia. APPROACH AND RESULTS: The angiogenic potential of miR-126-3p was tested in human umbilical vein endothelial cells in vitro. UTMD of miR-126-3p was tested in vivo in Fischer-344 rats before and after chronic left femoral artery ligation, evaluating target knockdown, miR-126-3p and miR-126-5p expression, phosphorylated Tie2 levels, microvascular perfusion, and vessel density. In vitro, miR-126-3p-transfected human umbilical vein endothelial cells showed repression of sprouty-related protein-1 and phosphatidylinositol-3-kinase regulatory subunit 2, negative regulators of vascular endothelial growth factor and angiopoietin-1 signaling, increased phosphorylated Tie2 mediated by knockdown of phosphatidylinositol-3-kinase regulatory subunit 2 and greater angiogenic potential mediated by both vascular endothelial growth factor/vascular endothelial growth factor R2 and angiopoietin-1 /Tie2 effects. UTMD of miR-126-3p resulted in targeted vascular transfection, peaking early after delivery and lasting for >3 days, and resulting in inhibition of sprouty-related protein-1 and phosphatidylinositol-3-kinase regulatory subunit 2, with minimal uptake in remote organs. Finally, UTMD of miR-126-3p to chronic ischemic hindlimb muscle resulted in improved perfusion, vessel density, enhanced arteriolar formation, pericyte coverage, and phosphorylated Tie2 levels, without affecting miR-126-5p or delta-like 1 homolog levels. CONCLUSIONS: UTMD of miR-126 results in improved tissue perfusion and vascular density in the setting of chronic ischemia by repressing sprouty-related protein-1 and phosphatidylinositol-3-kinase regulatory subunit 2 and enhancing vascular endothelial growth factor and angiopoietin-1 signaling, with no effect on miR-126-5p. UTMD is a promising platform for microRNA delivery, with applications for therapeutic angiogenesis.

The essential autophagy gene ATG7 modulates organ fibrosis via regulation of endothelial-to-mesenchymal transition.

Pulmonary fibrosis is a progressive disease characterized by fibroblast proliferation and excess deposition of collagen and other extracellular matrix components. Although the origin of fibroblasts is multifactorial, recent data implicate endothelial-to-mesenchymal transition as an important source of fibroblasts. We report herein that loss of the essential autophagy gene ATG7 in endothelial cells (ECs) leads to impaired autophagic flux accompanied by marked changes in EC architecture, loss of endothelial, and gain of mesenchymal markers consistent with endothelial-to-mesenchymal transition. Loss of ATG7 also up-regulates TGFß signaling and key pro-fibrotic genes in vitro. In vivo, EC-specific ATG7 knock-out mice exhibit a basal reduction in endothelial-specific markers and demonstrate an increased susceptibility to bleomycin-induced pulmonary fibrosis and collagen accumulation. Our findings help define the role of endothelial autophagy as a potential therapeutic target to limit organ fibrosis, a condition for which presently there are no effective available treatments.