Microvascular Endothelial Cell Tube Formation Research Articles

Stachyose with effect on anti-angiogenic activity from Salvia yunnanensis

Salvia yunnanensis is a traditional Chinese medicine for promoting blood circulation and eliminating blood stasis. Previous researches reported that Tanshinone IIA from Salvia yunnanensis had anti-angiogenic effect, however, whether its carbohydrates with similar effect is unknown. Here, we showed that a stachyose (Sta) from Salvia yunnanensis was purified by DEAE SepharoseTM Fast Flow anion-exchange chromatography and Bio-Gel P-2 gel permeation chromatography. Structure analyses suggested that Sta was tetrasaccharide as α-Gal-(1 → 6)-α-Gal-(1 → 6)-α-Glc-(1 → 2)-β-Fru. Further sulfated Sta was made and the degree of sulfate substitution was measured as 0.98. Bioactivity studies showed that Sta might block human microvascular endothelial cells tube formation in vitro and in vivo without significant cytotoxicity. Mechanism study suggested that Sta might bind to Galectin-3 (KD: 5.777E−6) and inhibit its expression to block angiogenesis linked to VEGFR/FAK and PDGFR/AKT/ERK signaling. Evidence also supported that Sta might be a potential compound for developing stachyose as an anti-angiogenesis functional food.

Sustained release of vascular endothelial growth factor A and basic fibroblast growth factor from nanofiber membranes reduces oxygen/glucose deprivation-induced injury to neurovascular units.

Upregulation of vascular endothelial growth factor A/basic fibroblast growth factor (VEGFA/bFGF) expression in the penumbra of cerebral ischemia can increase vascular volume, reduce lesion volume, and enhance neural cell proliferation and differentiation, thereby exerting neuroprotective effects. However, the beneficial effects of endogenous VEGFA/bFGF are limited as their expression is only transiently increased. In this study, we generated multilayered nanofiber membranes loaded with VEGFA/bFGF using layer-by-layer self-assembly and electrospinning techniques. We found that a membrane containing 10 layers had an ideal ultrastructure and could efficiently and stably release growth factors for more than 1 month. This 10-layered nanofiber membrane promoted brain microvascular endothelial cell tube formation and proliferation, inhibited neuronal apoptosis, upregulated the expression of tight junction proteins, and improved the viability of various cellular components of neurovascular units under conditions of oxygen/glucose deprivation. Furthermore, this nanofiber membrane decreased the expression of Janus kinase-2/signal transducer and activator of transcription-3 (JAK2/STAT3), Bax/Bcl-2, and cleaved caspase-3. Therefore, this nanofiber membrane exhibits a neuroprotective effect on oxygen/glucose-deprived neurovascular units by inhibiting the JAK2/STAT3 pathway.

Etanercept as a TNF-alpha inhibitor depresses experimental retinal neovascularization.

The formation of retinal neovascularization (RNV) is the primary pathological process underlying retinopathy of prematurity (ROP). Previous studies have shown that inflammatory factors are related to the formation of RNV. Tumor necrosis factor-α (TNF-α), as an important factor in the inflammatory response, is involved in the regulation of RNV formation. However, the mechanism through which TNF-α inhibition reduces RNV formation is not fully clarified. Therefore, the purpose of this study was to explore the effect of etanercept, an inhibitor of TNF-α, on RNV, and its possible mechanism. In vivo, an oxygen-induced retinopathy (OIR) mouse model was used to determine the effect of etanercept on the formation of RNV by performing immunostaining. The effect of etanercept on tumor necrosis factor receptor-associated factor 2 (TRAF2), pro-angiogenic-related factors, and pro/anti-inflammatory factors in OIR mice was assessed by real-time PCR and Western blotting. In vitro, the effect of etanercept on TNF-α-induced human retinal microvascular endothelial cell tube formation was evaluated by tube formation assays, and the potential mechanism of etanercept was explored by Western blotting. In vivo, etanercept reduced the area of RNV and decreased the expression of TRAF2 in the OIR mouse model. Etanercept also suppressed the expression of several pro-angiogenic factors and regulated the pro/anti-inflammatory factors. In vitro, etanercept reduced endothelial cell tube formation by inhibiting activation of the NF-κB signaling pathway. Etanercept can regulate pro/anti-inflammatory factors and reduce the expression of pro-angiogenic factors by inhibiting NF-κB phosphorylation, thereby reducing RNV formation.

Chondrocytes Promote Vascularization in Fracture Healing Through a FOXO1-Dependent Mechanism.

Chondrocytes play an essential role in fracture healing by producing cartilage, which forms an anlage for endochondral ossification that stabilizes the healing fracture callus. More recently it has been appreciated that chondrocytes have the capacity to produce factors that may affect the healing process. We examined the role of chondrocytes in angiogenesis during fracture healing and the role of the transcription factor forkhead box-O 1 (FOXO1), which upregulates wound healing in soft tissue. Closed fractures were induced in experimental mice with lineage-specific FOXO1 deletion by Cre recombinase under the control of a collagen-2α1 promoter element (Col2α1Cre+ FOXO1L/L ) and Cre recombinase negative control littermates containing flanking loxP sites (Col2α1Cre- FOXO1L/L ). Experimental mice had significantly reduced CD31+ new vessel formation. Deletion of FOXO1 in chondrocytes in vivo suppressed the expression of vascular endothelial growth factor-A (VEGFA) at both the protein and mRNA levels. Overexpression of FOXO1 in chondrocytes in vitro increased VEGFA mRNA levels and VEGFA transcriptional activity whereas silencing FOXO1 reduced it. Moreover, FOXO1 interacted directly with the VEGFA promoter and a deacetylated FOXO1 mutant enhanced VEGFA expression whereas an acetylated FOXO1 mutant did not. Lastly, FOXO1 knockdown by siRNA significantly reduced the capacity of chondrocytes to stimulate microvascular endothelial cell tube formation in vitro. The results indicate that chondrocytes play a key role in angiogenesis which is FOXO1 dependent and that FOXO1 in chondrocytes regulates a potent angiogenic factor, VEGFA. These studies provide new insight into fracture healing given the important role of vessel formation in the fracture repair process. © 2018 American Society for Bone and Mineral Research.

Photostable and Biocompatible Fluorescent Silicon Nanoparticles-Based Theranostic Probes for Simultaneous Imaging and Treatment of Ocular Neovascularization.

Ocular neovascularization can result in devastating diseases that lead to marked vision impairment and eventual visual loss. In clinical implementation, neovascular eye diseases are first diagnosed by fluorescein angiography and then treated by multiple intravitreal injections, which nevertheless involves vision-threatening complications, as well as lack of real-time monitoring disease progression and timely assessment of therapeutic outcomes. To address this critical issue, we herein present a kind of theranostic agents made of peptide-functionalized silicon nanoparticles (SiNPs), suitable for simultaneous ocular neovascularization imaging and therapy. Typically, in addition to negligible toxicity and high specific binding ability to human retinal microvascular endothelial cells tube formation, the cyclo-(Arg-Gly-Asp-d-Tyr-Cys) ( c-(RGDyC))-conjugated SiNPs (SiNPs-RGD) features efficacious antiangiogenic ability in wound healing migration, transwell migration, transwell invasion, and tube formation assays. Taking advantage of these unique merits, we further employ the SiNPs-RGD for labeling angiogenic blood vessels and neovascularization suppression, demonstrating obvious inhibition of new blood vessels formation in mouse corneas. These results suggest the SiNPs-RGD as a novel class of high-quality theranostic probes is suitable for simultaneous diagnosis and treatment in ocular neovascular diseases.

Rottlerin as a therapeutic approach in psoriasis: Evidence from in vitro and in vivo studies

Rottlerin is a natural polyphenolic compound that was initially indicated as a PKCδ inhibitor. However, it was recently revealed that it may target a number of molecules and have biological effects on various cell types and is considered as a possible agent for tumor and cell proliferative diseases. Psoriasis is a chronic inflammatory cutaneous disorder with undefined etiology and is characterized by abnormal cellular proliferation, angiogenesis, and inflammation. Therefore, this paper investigates the regulatory effects of rottlerin on normal human epidermal keratinocytes (NHEKs) and imiquimod (IMQ)-induced psoriasiform (IPI) lesions. In vitro results showed that rottlerin inhibited cell proliferation in NHEKs through growth arrest and NFκB inhibition. It may also induce apoptosis in an autophagy-dependent pathway. We found that rottlerin inhibited human microvascular endothelial cells tube formation on matrigel. Rottlerin also decreased the cell senescence of keratinocytes and intracellular ROS generation, which indicated its antioxidant effect. We also showed that rottlerin affects the expression of keratinocyte proliferation biomarkers. In 12-O-tetradecanoylphorbol13-acetate (TPA)–induced keratinocytes, rottlerin significantly inhibited the expression of the induced pro-inflammatory cytokines in keratinocytes. An animal experiment provided the corresponding evidence based on this evidence in vitro, by using IPI model, we found that rottlerin could relieve the psoriasiform of BALB/c mice by inhibiting keratinocyte proliferation, inflammatory cell infiltration, and vascular proliferation. In conclusion, our results suggest that rottlerin may prove useful in the development of therapeutic agents against psoriasis. However, the deep mechanism still requires further study.

Abstract 24: Hydrogen Sulfide Potentiates Bone Marrow-derived Angiogenic Progenitor Cell-mediated Ischemic Limb Angiogenesis in Diabetic Animals

Rationale: Critical hindlimb ischemia (CLI) represents an outcome of peripheral artery disease with a high incidence in diabetic patients. Recently, insufficient hydrogen sulfide (H 2 S) production has been implicated in cardiovascular disease. Objective: Explore the role of H 2 S in diabetes-induced bone marrow-derived angiogenic progenitor cells (BMAPC) dysfunction and its therapeutic effects on ischemic hindlimb injury of diabetes. Methods and Results: Diabetic BMAPCs from db/db mice showed decreased intracellular H 2 S production and blunted cystathionine γ-lyase (CSE) expression-an enzyme required for H 2 S generation. High glucose (HG) inhibited H 2 S production, migration and increased apoptosis of BMPAC that was rescued by H 2 S donor diallyl trisulfide (DATS) or overexpression of CSE. Administration DATS or local injection of diabetic BMAPCs overexpressing CSE significantly potentiated BMAPC-mediated blood flow recovery, capillary and arteriole formation, skeletal muscle architecture preservation, cell survival and decrease of perivascular infiltration of monocytes (CD68 + ) cells in ischemic skeletal muscle. Moreover, overexpression of CSE increased diabetic BMAPCs homing and engraftment in ischemic hindlimb. HG-impaired human cardiac microvascular endothelial cells (HCMVECs) tube formation and migration were rescued by DATS or overexpression of CSE. Mechanistically, HG increased threonine-495 phosphorylation of eNOS (eNOS-pT495) and inhibited nitric oxide (NO) production in HCMVECs which were rescued by DATS or overexpression of CSE. Silencing CSE by siRNA impaired tube formation and increased eNOS-pT495 expression in HCMVECs. Finally, HG-treated BMAPCs impaired mouse microvascular endothelial cell tube formation that was rescued by DATS. Conclusions: Our data suggests that CSE downregulation-induced H 2 S insufficient plays a critical role in diabetes-mediated BMAPC dysfunction. Administration of DATS or overexpression of CSE improves diabetic BMAPC-mediated angiogenesis/neovascularization via, at least partially, eNOS inactivation/NO reduction pathways. Our data indicate that H 2 S and overexpression of CSE in diabetic BMAPCs may open novel avenues for cell-based therapeutics of CLI in diabetic patients.

Abstract 329: Microparticles Induce Antiangiogenic Signaling in Microvascular Endothelial Cells by Activating a CD36-Dependent Signaling Pathway Involving Src Kinases and Reactive Oxygen Species

Cell-derived microparticles (MP), ≤1μm vesicles shed from vascular and blood cells during activation or apoptosis, circulate in plasma and are biomarkers of cardiovascular disease. The type 2 scavenger receptor CD36 binds MP via phosphatidylserine exposed on their surface. On microvascular endothelial cells (MVEC) CD36 induces anti-angiogenic signals via its cognate protein ligands, such as thrombospondin-1. We hypothesized that CD36-MP interactions on MVEC would inhibit angiogenesis. MP generated in vitro from THP1 cells dose dependently inhibited MVEC tube formation in in vitro matrigel angiogenesis assays (>50% inhibition of number of tubes formed, branch points, and segments; p<0.05). MP derived from human umbilical vein endothelial cells (HUVEC), MVEC and erythrocytes also inhibited tube formation showing that the anti-angiogenic effect of MP is independent of their cell of origin. THP1 MP also inhibited MVEC migration in a transwell assay by 80% (p<0.001). Early passage MVEC with high CD36 expression were inhibited more than late passage MVEC with low CD36 expression (80% vs 33%; p<0.001) establishing the specificity of CD36 in this process. Further, in HUVEC that do not express CD36, inhibition of migration by THP1 MP was only 44% (p<0.001) but lentiviral-mediated heterologous expression of murine CD36 in HUVEC restored inhibition of migration to levels similar to that of MVEC (82%; p<0.001), corroborating the specificity of CD36 in MP mediated inhibition of migration. Since CD36 dependent generation of reactive oxygen species (ROS) is necessary to inhibit cell migration in macrophages, we tested if MP can induce ROS in MVEC. THP1 MP dose dependently induced ROS in MVEC by two fold compared to control at 1hr (p<0.05). NADPH oxidase inhibitor, apocynin, blocked MP induced ROS induction by >95% and partially (33%) reversed the inhibition of tube formation in MVEC by THP1 MP (p<0.05). Western blots of THP1 MP treated MVEC showed time-dependent activation of Fyn kinase, and PP2, a Src family kinase inhibitor, blocked MP induced ROS induction by >95%. These studies identify a novel CD36 dependent mechanistic pathway through which cell-derived MP inhibit MVEC angiogenic activities through ROS generation involving NADPH oxidase and Src kinase Fyn.

Molecular Basis of Antiangiogenic Thrombospondin-1 Type 1 Repeat Domain Interactions With CD36

Antiangiogenic activity of thrombospondin-1 and related proteins is mediated by interactions between thrombospondin type 1 repeat (TSR) domains and the CD36, LIMP-2, Emp sequence homology (CLESH) domain of the endothelial cell receptor CD36. We sought to characterize key molecular determinants of the interaction between thrombospondin-1 TSR domains and the CD36 CLESH domain. Recombinant thrombospondin-1 TSR2 and TSR(2,3) constructs inhibited microvascular endothelial cell migration, microvascular endothelial cell tube formation, and vessel sprouting in aortic ring assays. Interaction with CD36 CLESH decoy peptides negated these effects. Mutational analyses identified a cluster of residues that confer positive charge to the TSR2 surface and mediate interaction with CD36 CLESH. Antiangiogenic activity was significantly reduced by charge-neutralizing mutations of the Arg-Trp ladder in TSR2, but not TSR3. Additionally, I438 and K464 of TSR2 were shown to be required for CD36 CLESH binding to TSR2. A complementary acidic cluster within CD36 CLESH is also required for antiangiogenic activity. Thrombospondin-1 interacts with CD36 CLESH through electrostatic interactions mediated by a positively charged TSR2 surface and multiple negatively charged CD36 CLESH residues. Two key residues serve as specificity determinants that identify other TSR domains that interact with CD36 CLESH.

Mechanisms of vascular injury in diabetes: lessons from vascular cells in culture and transgenic animals

Microvessels are composed of two cell types, endothelial cells (EC) and pericytes. The decrease in the latter (pericyte loss) and the increase in the former (angiogenesis) are the hallmarks of diabetic retinopathy. Coculture experiments revealed that the growth and function of EC are under a control of neighboring pericytes, thus explaining why endothelial derangement follows pericyte loss. During prolonged hyperglycemic exposure, nonenzymatically glycated protein derivatives termed advanced glycation endoproducts (AGE) are formed at an accelerated rate and accumulate in palsma and various tissues. When bovine retinal pericytes were cultured with AGE-bovine serum albumin, the number of viable pericytes was decreased, which was abolished by a DNA complement of mRNA coding for a cell-surface receptor for AGE (RAGE). AGE can act also on EC, again in a RAGE-dependent manner. They stimulated the proliferation and tube formation of microvascular EC, and inhibited prostacyclin synthesis but induced plasminogen activator inhibitor-1. Thus, AGE predispose to both angiogenesis and thrombogenesis. The angiogenic activity of AGE was found to be mediated by autocrine vascular endothelial growth factor. To address the role of RAGE in the development of diabetic pathology, we created transgenic mice that overexpress RAGE in vascular cells and made them diabetic by crossbreeding with another transgenic line that develops insulin-dependent diabetes early after birth. The resultant double transgenic mice exhibited accelerated nephropathy as evidenced by significantly higher scores in albuminuria, glomerulosclerosis and serum creatinine compared with single transgenic and nontransgenic littermates. The nephropathy was ameliorated by the administration of an inhibitor of AGE formation. Indices diagnostic of diabetic retinopathy were also prominent in the double transgenic mouse model. Further, the regulators of RAGE gene expression have been identified. Transcription of human RAGE gene was induced by tumor necrosis factor-α and AGE ligands themselves through nuclear factor-κB activation, and by 17β-estradiol through Sp-1. The results thus suggest that the AGE-RAGE system plays an active role in the development of diabetic vasculopathy and is a promising target in the prophylaxis and therapy of this disease. Biomedical Reviews 2000; 11:19-27.

Cerebral microvascular endothelial cell tube formation: role of astrocytic epoxyeicosatrienoic acid release.

Cerebral microvascular endothelial cells (CMVEC) form tubes when cocultured with astrocytes (AS). Therefore, it appears that AS may be important in mediating angiogenesis in the brain. We hypothesized that AS modulate CMVEC tube formation by releasing a soluble factor. Thymidine incorporation in cultured CMVEC increased 305% when incubated with 50% conditioned AS medium for 24 h [control: 52,755 +/- 4,838 counts per minute (cpm) per well, conditioned 161,082 +/- 12,099 cpm/well, n = 8]. Because our laboratory has previously shown that AS can produce epoxyeicosatrienoic acids (EETs), which are known mitogens, we investigated whether release of EETs by AS is responsible for tube formation in the CMVEC-AS coculture. AS were seeded on Lab-Tek slides, CMVEC were seeded on the AS the next day, and cultures were allowed to progress for another 5 days with and without cytochrome P-450 epoxygenase blockade by 17-octadecynoic acid (17-ODYA). Tube formation in cocultures receiving 17-ODYA was significantly inhibited compared with control (93.8%). These data suggest that tube formation requires the release of EETs by AS.