Sex-specific angiogenic responses in endothelial cells—role of the pluripotency factor OCT4

Redox signaling mediated by Nox2-containing NADPH oxidase has been implicated in angiogenic responses both in vitro and in vivo. Because Nox4 type NADPH oxidase is also highly expressed in endothelial cells, we studied the role of Nox4 in angiogenic responses in human endothelial cells in culture. Inhibition of Nox4 expression by small interfering RNA reduced angiogenic responses as assessed by the tube formation and wound healing assays, in both human microvascular and umbilical vein endothelial cells. Overexpression of wild-type Nox4 enhanced, whereas expression of a dominant negative form of Nox4 suppressed the angiogenic responses in endothelial cells. These effects were mimicked by exogenous H2O2 and the antioxidant compound ebselen, respectively. Overexpression of Nox4 enhanced receptor tyrosine kinase phosphorylation and the activation of extracellular signal-regulated kinase (Erk). Inhibition of the Erk pathway reduced the endothelial angiogenic responses. Nox4 expression also promotes proliferation and migration of endothelial cells, and reduced serum deprivation-induced apoptosis. Nox4 type NADPH oxidase promotes endothelial angiogenic responses, at least partly, via enhanced activation of receptor tyrosine kinases and the downstream Erk pathway.

Editor's evaluation: The effects of caloric restriction on adipose tissue and metabolic health are sex- and age-dependent

NOX2, NOX4, and mitochondrial-derived reactive oxygen species contribute to angiopoietin-1 signaling and angiogenic responses in endothelial cells

Boosting angiogenesis is a crucial process to enhance tissue growth in tissue engineering (TE). Hepatoma-derived growth factor (HDGF) has been identified as an angiogenic factor, but its involvement in angiogenesis in an arteriovenous loop-based TE chamber developed by the laboratory is unclear. In this study, the authors first examined the effects of HDGF on angiogenic responses in endothelial cells and in a corneal model of neovascularization, and then characterized the expression of HDGF in the TE chamber. HDGF (1–500 ng/mL) induced concentration-dependent angiogenic responses in human endothelial cells in vitro (proliferation, migration, and tube formation). Local application of HDGF stimulated neovascularization in a rat model of corneal angiogenesis. In the TE chamber, there was an increase in blood vessel volume from day 3 to day 14. Immunofluorescence microscopy revealed that HDGF is highly expressed in the neovessels in the chamber. Peak expression of HDGF (day 3) coincided with the infiltration of inflammatory cells, and the mRNA level of endogenous HDGF correlated with that of tumor necrosis factor α (TNFα). In vitro, TNFα stimulated HDGF expression in endothelial cells. The data suggest that HDGF may be involved in angiogenic responses in the TE chamber and the proinflammatory cytokine TNFα may have a pivotal role in stimulating HDGF expression. Enhancing HDGF signaling may be a new approach to extend vascularization for TE.

Sexually Dimorphic Patterns of Episomal rAAV Genome Persistence in the Adult Mouse Liver and Correlation With Hepatocellular Proliferation

Background: Peripheral artery disease (PAD) is a highly prevalent vascular occlusive disease which mostly affects the lower limb. Emerging studies proved the critical functions of endothelial cells in arteriogenesis and ischemic limb blood flow recovery. However, very few targets with endothelial cell exclusivity have been identified. Here, we explored the role of endothelial BCL-2 associated athanogene 3 (Bag3) in ischemic limb recovery. Hypothesis: We hypothesized that the loss of endothelial specific BAG3 would attenuate blood flow and muscle recovery following HLI. Methods: Hind limb ischemia (HLI) was induced in Bag3 Cdh5(PAC)-CreERT2 male and female mice after tamoxifen (2mg/day x 5 days) or vehicle treatment ( n ≥12/group). Limb blood flow was measured by laser Doppler perfusion imaging (LDPI) and isometric muscle force production, vascular density and muscle morphology were assessed after HLI. Results: LDPI analysis revealed no differences in the blood flow between groups at HLI d7 ( n=9 ; day 7, 21.86% ischemic/non-ischemic ± 3.80% vs 18.05% ± 1.96% SEM; p=0.05). There was no deficit in isometric force recovery of the EDL ( n ≥12; 57.76 ± 15.92 vs 83.05 ± 27.12 SEM; p =0.05) in KO animals at HLI d7. Similar rates of CD31+ vessel disorganization was observed in both groups at HLI d7, ( n =7; 4626.0 um 2 CD31+ area ± 869 vs 4870.9 ± 1056 SEM) consistent with recovering ischemic tissue. There were also no differences in blood flow recovery assessed by LDPI at HLI d28 ( n =7; 50.74% ischemic/non-ischemic ± 7.4% vs 59.17 ± 8.21; n=9). Conclusions: Endothelial cell specific Bag3 knockout did not impair vascular morphology or perfusion recovery at baseline or following HLI in a murine model of peripheral artery disease. No differences in muscle morphology or contractile function at baseline or after HLI at either timepoint were observed. In summary, we reveal an unexpectedly minor role of endothelial cell BAG3 in tissue recovery following ischemic injury.

Vascular Gene Transfer of SDF-1 Promotes Endothelial Progenitor Cell Engraftment and Enhances Angiogenesis in Ischemic Muscle

The sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) is key to Ca(2+) homeostasis and is redox-regulated by reversible glutathione (GSH) adducts on the cysteine (C) 674 thiol that stimulate Ca(2+) uptake activity and endothelial cell angiogenic responses in vitro. We found that mouse hind limb muscle ischemia induced S-glutathione adducts on SERCA in both whole muscle tissue and endothelial cells. To determine the role of S-glutathiolation, we used a SERCA 2 C674S heterozygote knock-in (SKI) mouse lacking half the key thiol. Following hind limb ischemia, SKI animals had decreased SERCA S-glutathione adducts and impaired blood flow recovery. We studied SKI microvascular endothelial cells in which total SERCA 2 expression was unchanged. Cultured SKI microvascular endothelial cells showed impaired migration and network formation compared with wild type (WT). Ca(2+) studies showed decreased nitric oxide (·NO)-induced (45)Ca(2+) uptake into the endoplasmic reticulum (ER) of SKI cells, while Fura-2 studies revealed lower Ca(2+) stores and decreased vascular endothelial growth factor (VEGF)- and ·NO-induced Ca(2+) influx. Adenoviral overexpression of calreticulin, an ER Ca(2+) binding protein, increased ionomycin-releasable stores, VEGF-induced Ca(2+) influx and endothelial cell migration. Taken together, these data indicate that the redox-sensitive Cys-674 thiol on SERCA 2 is required for normal endothelial cell Ca(2+) homeostasis and ischemia-induced angiogenic responses, revealing a novel redox control of angiogenesis via Ca(2+) stores.

ELR+ CXC chemokines are potent promoters of angiogenesis and have been shown to induce chemotaxis in endothelial cells (ECs). We reported previously that EC chemotaxis was markedly enhanced in systemic arterial ECs compared to pulmonary artery ECs in response to MIP-2, a potent proangiogenic ELR+ CXC chemokine ligand, which binds the G-protein coupled receptor, CXCR2. Also, we previously showed equivalent levels of CXCR2 expression in the two EC subtypes. However, differences in EC motility in response to MIP-2 may depend on differential utilization of signaling pathways. We studied the roles of protein kinase B/AKT and small GTPase RhoA in MIP-2 activation of mouse aortic (n=4) and pulmonary artery (n= 3) ECs. Additionally, since tissue hypoxia increases the angiogenic potential of systemic ECs, we studied ECs after hypoxia (24hrs)/reoxygenation (48hrs). MIP-2 (30min, 10 ng/ml) induced a greater increase in AKT phosphorylation (pAKT) in aortic ECs (33%) compared to pulmonary artery ECs (17%). Hypoxia/reoxygenation increased pAKT in aortic ECs (46%) but did not alter pulmonary artery EC pAKT. Only aortic ECs showed an increase in RhoA activation (40%) after MIP-2 treatment and hypoxia/ reoxygenation further increased RhoA activation after MIP-2 in aortic ECs (71%). These results confirm that AKT and RhoA are involved in MIP-2 induced activation of a proangiogenic phenotype, preferentially in aortic ECs. Supported by HL71605

Background Despite similar rates of obesity, the prevalence and severity of obesity‐related metabolic diseases is lower in females compared to males. This relates with better preserved function of the adipose tissue in females. Consistent with the importance of capillaries in maintaining tissue functions, insufficient vascularization in expanding adipose tissue leads to adipocyte dysfunction, inflammation and whole‐body metabolic disturbances. Previously, we reported that angiogenesis is higher in the perigonadal adipose tissue in female compared to male mice on a high‐fat diet, leading to a higher capillary density in female adipose. Endothelial cells line capillaries and coordinate different cellular processes involved in angiogenesis, allowing for the necessary vascular remodelling needed in adipose maintenance. We hypothesize that differential gene networks/pathways in the endothelial cells of females compared to males support the better angiogenic response in female adipose tissue. Methods Male and female (5‐7 weeks old; n=4/group) mice were fed a high‐fat diet (60% kcal fat) for 7 weeks. White adipose tissue was harvested and used for whole‐mount imaging and endothelial cell isolation. Adipose tissue was stained with lectin and imaged to determine the vascular density. RNA extracted from the isolated endothelial cells was analyzed by RNA‐sequencing. Transcripts were assessed for differentially expressed genes (DEGs). Gene ontologies and gene set enrichment analysis were used to identify patterns of gene expression. Results Adipose tissue showed greater vascularization in high fat ‐fed females with vascular areas 27.2 ± 2.2 and 21.0 ± 0.6 % field of view in females and males respectively. RNA‐sequencing led to identification of 1225 DEG between male and female endothelial cells (P<0.01). Ontology analyses of the DEGs showed that male endothelial cells expressed higher levels of genes enriched in inflammatory responses and immune processes including chemokine production, complement/coagulation and leukocyte adhesion. In contrast, female endothelial cells lacked an inflammatory signature. Instead, the genes that were higher in female endothelial cells were related to cellular dynamics, such as machinery regulating transcription, translation, DNA replication and axon guidance. Conclusion Adipose endothelial cells from male and female mice have a high number of DEGs that reflect differences in endothelial cell angiogenic phenotype as well as other unique profiles that may be relevant to adipose function. Upregulation of ontologies relating to cellular replication and protein synthesis in females supports the observation of a better angiogenic response in the female adipose tissue and implies that intrinsic differences in male and female endothelial cells contribute to this capacity.

Atherosclerosis is now viewed as an inflammatory disease occurring preferentially in arterial regions exposed to disturbed flow conditions, including oscillatory shear stress (OS), in branched arteries. In contrast, the arterial regions exposed to laminar shear (LS) are relatively lesion-free. The mechanisms underlying the opposite effects of OS and LS on the inflammatory and atherogenic processes are not clearly understood. Here, through DNA microarrays, protein expression, and functional studies, we identify bone morphogenic protein 4 (BMP4) as a mechanosensitive and pro-inflammatory gene product. Exposing endothelial cells to OS increased BMP4 protein expression, whereas LS decreased it. In addition, we found BMP4 expression only in the selective patches of endothelial cells overlying foam cell lesions in human coronary arteries. The same endothelial patches also expressed higher levels of intercellular cell adhesion molecule-1 (ICAM-1) protein compared with those of non-diseased areas. Functionally, we show that OS and BMP4 induced ICAM-1 expression and monocyte adhesion by a NFkappaB-dependent mechanism. We suggest that BMP4 is a mechanosensitive, inflammatory factor playing a critical role in early steps of atherogenesis in the lesion-prone areas.

Fatty acid-binding proteins (FABP) are small molecular mass intracellular lipid chaperones that are expressed in a tissue-specific manner with some overlaps. FABP4 and FABP5 share ~55% amino acid sequence homology and demonstrate synergistic effects in regulation of metabolic and inflammatory responses in adipocytes and macrophages. Recent studies have shown that FABP4 and FABP5 are also co-expressed in a subset of endothelial cells (EC). FABP4, which has a primarily microvascular distribution, enhances angiogenic responses of ECs, including proliferation, migration, and survival. However, the vascular expression of FABP5 has not been well characterized, and the role of FABP5 in regulation of angiogenic responses in ECs has not been studied to date. Herein we report that while FABP4 and FABP5 are co-expressed in microvascular ECs in several tissues, FABP5 expression is also detected in ECs of larger blood vessels. In contrast to FABP4, EC-FABP5 levels are not induced by VEGF-A or bFGF. FABP5 deficiency leads to a profound impairment in EC proliferation and chemotactic migration. These effects are recapitulated in an ex vivo assay of angiogenesis, the aortic ring assay. Interestingly, in contrast to FABP4-deficient ECs, FABP5-deficient ECs are significantly more resistant to apoptotic cell death. The effect of FABP5 on EC proliferation and survival is mediated, only in part, by PPARδ-dependent pathways. Collectively, these findings demonstrate that EC-FABP5, similar to EC-FABP4, promotes angiogenic responses under certain conditions, but it can also exert opposing effects on EC survival as compared to EC-FABP4. Thus, the balance between FABP4 and FABP5 in ECs may be important in regulation of angiogenic versus quiescent phenotypes in blood vessels.

Angiogenesis and vasculogenesis: Inducing the growth of new blood vessels and wound healing by stimulation of bone marrow–derived progenitor cell mobilization and homing

DYRK1A Kinase Positively Regulates Angiogenic Responses in Endothelial Cells

Introduction: In addition to aerobic exercise, it has been recognized that resistance exercise has beneficial effects for the patients with peripheral arterial disease. Recently, muscle-derived exosomes are demonstrated to convey many types of signaling molecules including microRNAs (miRNAs). Hypothesis: We assessed the hypothesis that exosomal miRNA secreted by growing muscle promotes angiogenic response in endothelial cells. Methods: We utilized skeletal muscle-specific inducible Akt1 transgenic (Akt1-TG) mice that can induce growth of skeletal muscles without exercise training. Exosomes was purified combined usage of ultracentrifugation, and miRNAs were extracted from purified exosomes by spin column-based method. Real-time PCR array and droplet digital PCR was performed to evaluate exosomal miRNA expression. Results: Akt1-TG mice showed remarkable skeletal muscle growth 2 weeks after gene activation (weight of gastrocnemius muscle: 0.21±0.03 vs 0.16±0.02, g, p<0.01). The amount of exosomal proteins did not different between Akt1-TG mice and WT mice (977.7±11.3 vs 826.0±45.2, μg/mL serum, p=0.07). KEGG pathway frequency analysis for 4665 target genes identified by real-time PCR array of miRNAs revealed a significant increase in Akt and its downstream signaling pathways. Among upregulated miRNAs, droplet digital PCR identified that miR-1, -133 and -206 expression was significantly upregulated in Akt1 TG mice compared with WT mice in serum. miR206 was increased in IGF-1-stimulated myotubes. PKH26-labeled exosomes were taken up by human umbilical vein endothelial cells. Exogenous supplementation of exosomal miRNA206 promoted angiogenesis as revealed by spheroid assay, and increased expression of HIF-1 and VEGF in endometrial cells. Conclusions: Exosomal miR206 was upregulated in blood stream in Akt1-TG mice and IGF-stimulated cultured myotubes. Exogenous supplementation of miR206 promoted angiogenic response in endothelial cells. Our data suggest that miR206 secreted from growing muscle act on endothelial cells and promote angiogenesis.