Migration Of Microvascular Endothelial Cells Research Articles

Simultaneous interference of SP1 and HIF1α retarding the proliferation, migration, and invasion of human microvascular endothelial cells (HMEC-1) under hypoxia.

To investigate the regulation of special protein 1 (SP1) and hypoxia-inducible factor-1α (HIF1α) on human microvascular endothelial cells (HMEC-1) under hypoxic conditions. The expression of SP1 and HIF1α under normoxia and hypoxic conditions were assessed by Western blot. SP1 and HIF1α were knocked down by small interfering RNA (siRNA) under hypoxic conditions. The proliferation, migration, and invasion of HMEC-1 were measured by cell counting kit 8, 5-ethynyl-2'-deoxyuridine and Transwell coculture system. Western blot analysis and Immunofluorescence were carried out to study the mechanisms of simultaneously inhibiting the adenosine triphosphatase (CD39), 5'-nucleotidase (CD73), adenosine, and vascular endothelial growth factor (VEGF). We compared the inhibitory effects between groups concurrently interfering SP1, HIF-1α, and ranibizumab under hypoxic conditions. Under hypoxic conditions, the protein expression of SP1 and HIF1α was increased in HMEC-1, contrarily, SP1 siRNA and HIF1α siRNA downregulated the expression. Simultaneous inhibition of SP1 and HIF1α demonstrated a much greater restraint of proliferation, migration, and invasion characteristics on HMEC-1 than respectively knocking down SP1 or HIF1α and anti-VEGF drugs (0.5 mg/mL ranibizumab) (siRNA and the VEGF inhibitor were applied separately in different groups). Meanwhile, simultaneous inhibition of SP1 and HIF1α effectively reduced the expression of CD39, CD73, adenosine, and VEGF on HMEC-1 under hypoxic conditions. Our study demonstrated that both SP1 and HIF1α played important roles in HMEC-1 under hypoxia condition. Simultaneous inhibition of SP1 and HIF1α effectively decreased the activity of HMEC-1 under hypoxic conditions through the CD39-CD73-adenosine and VEGF angiogenesis pathways. Our study may provide a new approach to the treatment of retinal neovascular diseases.

O- glycosylation can regulate the proliferation and migration of human retinal microvascular endothelial cells through ZFR in high glucose condition

PurposeAngiogenesis is an essential part of the diabetes retinopathy (DR) process, and Zinc Finger RNA Binding Protein (ZFR) is important for vascularization to occur. However, the function and regulation of ZFR in DR development are not well understood. We hypothesized that high glucose condition could result in ZFR up-regulation in human retinal microvascular endothelial cells (HRMECs), thus contributing to disease progression, and O-glycosylation may be participated in this regulation. MethodsRetinas were harvested from streptozotocin (STZ)-induced rat model of diabetes. Human umbilical vein endothelial cells (HUVECs) and HRMECs cultured in high glucose concentration, and retinal tissues were detected for ZFR expression. We examined the role of ZFR on vasculogenic processes including proliferation and migration in the cell model of DR. The effect of O-glycosylation modification on ZFR was further assessed in HRMECs. ResultsExpression of ZFR was up-regulated in high glucose condition both in vitro and in vivo. ZFR induced proliferation and migration of HRMECs. Inhibition of O-glycosylation modification attenuated the expression of ZFR. ConclusionZFR plays an important role in the pathogenesis of DR, and its mechanism may through the modification of O-glycosylation. Our research suggests that ZFR may be used as a potential prognostic marker or potential therapeutic target for DR.

Downregulation of circRNA DMNT3B Contributes to Diabetic Retinal Vascular Dysfunction Through Targeting miR-20b-5p and BAMBI

Background: Diabetic retinopathy, a vascular complication of diabetes mellitus, is the leading cause of visual impairment and blindness. circRNAs act as competing endogenous RNA, sponging target miRNA and thus influencing mRNA expression in vascular diseases. We investigated whether and how circDNMT3B is involved in retinal vascular dysfunction under diabetic conditions. Methods: qRT-PCR was performed to detect expression of circDNMT3B, miR-20b-5p, and BAMBI in retinal microvascular endothelial cells under diabetic conditions. Western blot, Cell Counting Kit-8, Transwell, Matrigel tube formation, and retinal trypsin digestion assays were conducted to explore the roles of circDNMT3B/miR-20b-5p/BAMBI in retinal vascular dysfunction. Bioinformatics analysis and luciferase reporter, siRNA, and overexpression assays were used to reveal the mechanisms of the circDNMT3B/miR-20b-5p/BAMBI interaction. Electroretinograms were used to evaluate visual function. Findings: Upregulation of miR-20b-5p under diabetic conditions promoted proliferation, migration, and tube formation of human retinal microvascular endothelial cells (HRMECs), which was mediated by downregulated BAMBI. Under diabetic conditions, circDNMT3B, which acts as a sponge of miR-20b-5p, is downregulated. circDNMT3B overexpression reduced retinal acellular capillary number and alleviated visual damage in diabetic rats. Changes in expression of circDNMT3B and miR-20b-5p were confirmed in the proliferative fibrovascular membranes of patients with diabetic retinopathy. Interpretation: Downregulation of circDNMT3B contributes to vascular dysfunction in diabetic retinas through regulating miR-20b-5p and BAMBI, providing a potential treatment strategy for diabetic retinopathy. Funding Statement: This work was supported by the National Natural Science Foundation of China (No. 81790642, 31800872, 81570854, and 81770944), the National Key Basic Research Program of China (2013CB967503), the Shanghai Municipal Science and Technology Major Project (No.2018SHZDZX01) and ZJLab. Declaration of Interests: All authors declare no conflict interests. Ethics Approval Statement: All procedures were explained to patients, and informed consent was provided. All procedures met the ethical standards of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the guidelines of Fudan University. This study was approved by the Institutional Ethics Review Committee of the Eye & ENT Hospital of Fudan University.

The TrkB-T1 receptor mediates BDNF-induced migration of aged cardiac microvascular endothelial cells by recruiting Willin.

The mechanism of age‐related decline in the angiogenic potential of the myocardium is not yet fully understood. Our previous report revealed that the aging of cardiac microvascular endothelial cells (CMECs) led to changes in their expression of receptor Trk isoforms: among the three isoforms (TrkB‐FL, TrkB‐T1 and TrkB‐T2), only the truncated TrkB‐T1 isoform continued to be expressed in aged CMECs, which led to decreased migration of CMECs in aging hearts. Thus far, how BDNF induces signalling through the truncated TrkB‐T1 isoform in aged CMECs remains unclear. Here, we first demonstrated that aged CMECs utilize BDNF–TrkB‐T1 signalling to recruit Willin as a downstream effector to further activate the Hippo pathway, which then promotes migration. These findings suggest that the aging process shifts the phenotype of aged CMECs that express TrkB‐T1 receptors by transducing BDNF signals via the BDNF–TrkB‐T1–Willin–Hippo pathway and that this change might be an important mechanism and therapeutic target of the dysfunctional cardiac angiogenesis observed in aged hearts.

Cyclooxygenase-2 regulates HPS patient serum induced-directional collective HPMVEC migration via PKC/Rac signaling pathway

Hepatopulmonary syndrome (HPS) is a serious complication in patients with advanced liver disease. The pathological pulmonary angiogenesis contributes to the progression of HPS. Importantly, directional collective migration of endothelial cells is a critical event for pathological angiogenesis. Previously, we have demonstrated that the over-expression of Cyclooxygenase-2 (COX-2) was an important factor in the experimental HPS. However, the role of COX-2 in the directional collective migration of human pulmonary microvascular endothelial cells (HPMVECs) is unclear. Our study aims to evaluate the potential effect of COX-2 in the directional collective migration of HPMVECs under the stimulation of HPS patient serum. In this study, 9 patients with stable liver cirrhosis were screened for presence of HPS. We confirmed that HPS patient serum dramatically promoted the directional collective migration and angiogenesis of HPMVECs, while the COX-2 selective antagonist parecoxib significantly inhibited the directional collective migration of HPMVEC under the stimulation of HPS patient serum. In addition, HPS patient serum significantly upregulated the phosphorylation of PKC and promoted the activation of Rac via COX-2/PGE2 signaling pathway. Notably, silencing PKC activation attenuated the directional collective migration of HPMVEC induced by HPS patient serum. In conclusion, these results indicate that PKC/Rac signaling induced by COX-2 modulates collective directional migration of HPMVEC during pathological pulmonary angiogenesis in HPS patients.

Sulfated polysaccharide JCS1S2 inhibits angiogenesis via targeting VEGFR2/VEGF and blocking VEGFR2/Erk/VEGF signaling

More and more evidences suggested that sulfated natural glycans had impact on angiogenesis. However, the molecular targets and functional mechanism of glycans are still vague. JCS1S2 was the sulfated mannoglucan featured with a backbone of 1, 4-linked β-Manp and 1, 4-linked α-Glcp with sulfation at C-6 of β-Manp and α-Glcp residues, respectively. The degree of substitution of this sulfated polysaccharide was 1.74 and its molecular weight was 56.2 kDa. We provided evidences that JCS1S2 could disrupt angiogenesis both in vitro and in vivo. This sulfated polysaccharide inhibited migration and tube formation of human microvascular endothelial cells (HMEC-1) whereas showed no effect on the cells proliferation. Further study uncovered that JCS1S2 bound to both VEGF (vascular endothelial growth factor) (KD value: 4.82 × 10−9) and VEGFR2 (Vascular Endothelial Growth Factor Receptor 2) (KD value: 1.50 × 10-7) to inactivate VEGFR2 phosphorylation. In addition, JCS1S2 blocked downstream signaling and impaired the expression of VEGF and its transcription factor AP-1 (Activator protein-1). These results demonstrated that JCS1S2 interrupted angiogenesis via blocking VEGF signaling transduction and could be a potential anti-angiogenetic agent for disease treatment.

Inhibiting lncRNA ROR suppresses growth, migration and angiogenesis in microvascular endothelial cells by up-regulating miR-26.

Lower extremity arteriosclerosis is one of leading causes of death worldwide. Arteriosclerosis is closely related to microvascular endothelial cells. This study was aimed to explore the role of long non-coding RNA ROR in regulations of growth, migration, and angiogenesis of microvascular endothelial cells. Angiogenesis was determined by the number of tube-like cells on a matrigel extracellular matrix. Cell viability, apoptosis, and migration were determined by CCK-8 assay, PI/FITC-Annexin V staining method, and transwell assay, respectively. Relative RNA expression of ROR, miR-26, and angiogenesis-associated genes were analyzed by qRT-PCR. The protein expression of apoptosis- and angiogenesis-associated genes, as well as main factors in NF-κB and JAK1/STAT3 pathways, were analyzed by Western blot. LncRNA ROR silence inhibited viability, migration, and angiogenesis of HMEC-1 cells but promoted apoptosis of them. miR-26 expression was promoted after knocking down ROR expression. miR-26 overexpression enhanced the inhibitory effects of ROR silence on growth, migration, and angiogenesis in HMEC-1 cells, whereas, miR-26 silence impaired the effects of ROR silence. Finally, we found that NF-κB and JAK1/STAT3 signaling pathways were inhibited by ROR down-regulation. Similarly, miR-26 overexpression enhanced the inhibitory effect of ROR down-regulation on the pathways and miR-26 inhibition abrogated it. Down-regulating lncRNA ROR inhibited growth, migration and angiogenesis of microvascular endothelial cells possibly through up-regulation of miR-26. During this process, the activations of NF-κB and JAK1/STAT3 pathways were inhibited after interaction of ROR and miR-26.

DPP-4 inhibitors promote proliferation and migration of rat brain microvascular endothelial cells under hypoxic/high-glucose conditions, potentially through the SIRT1/HIF-1/VEGF pathway.

Vascular disease in diabetes, for example, stroke, presents a significant public health burden. Recently, the dipeptidyl peptidase 4 (DPP-4) inhibitor linagliptin has been found to counteract stroke among diabetic patients, showing great promise in drug repurposing and indication expansion. However, the molecular basis of this protection mechanism remains unknown. The expression and localization of DPP-4 in rat brain microvascular endothelial cells (rBMVECs) were assessed with immunofluorescent staining and Western blotting. The effects of DPP-4 inhibitors on cell proliferation and migration of rBMVECs were determined using MTT and transwell assays, separately. The influence of DPP-4 inhibition on the expression of molecular markers (eg, VEGF, eNOS, HIF-1α. SIRT1) was examined at both mRNA and protein levels with qRT-PCR and Western blotting, individually. DPP-4 inhibitors (40nmol/L linagliptin, 30μmol/L berberine) offer protection from hypoxia/high glucose induced impairments in the proliferation and migration of rBMVECs. Treatment with DPP-4 inhibitors counteracted the attenuating effects of hypoxic/high-glucose conditions on the expression of VEGF, eNOS, HIF-1α, and SIRT1, which can be completely eliminated by the inhibition of SIRT1 with 1mmol/L nicotinamide. The protection of rBMVECs from hypoxia/high-glucose induced impairment by DPP-4 inhibitors may be mediated by the SIRT1/HIF-1α/VEGF pathway.

Abstract 460: Diabetes Impairs Reparative Property of Bone Marrow-derived Endothelial Progenitor Cells: Role of Mir-499-mediated Hydrogen Sulfide Deficiency

Background: miR-499 has been considered as a potential biomarker for myocardial infarction. Overexpression of miR-499 impaired heart function and induced cardiac hypertrophy in mice. Recent studies suggested miR-499 suppresses tumor growth through the inhibition of vascular endothelial growth factor (VEGF) mediated angiogenesis. Furthermore, emerging evidences indicate that miR-499 is increased in heart of diabetic rodents and patients. Here, we tested the hypotheses that miR-499 impairs angiogenic ability of endothelial cells (ECs) under diabetic conditions. Methods and Results: miR-499 levels were examined in endothelial progenitor cells (EPCs), microvascular endothelial cells (MVECs) and plasma in diabetic db/db mice (8-week, male). Gender- and age-matched non-diabetic db/+ mice served as controls. EPCs were isolated from bone marrow density-gradient centrifugation. MVECs were isolated using collagenase. We found that miR-499 levels were significantly increased in EPCs, MVECs as well as plasma of db/db mice. miR-499 antagomiR rescued high glucose (25 mM D-glucose for 48 h)-impaired tube formation and migration of human cardiac microvascular endothelial cells (HCMVECs). Mechanistically, by luciferase assay, we found that miR-499 directly targets cystathionine γ-lyase (CSE), a major hydrogen sulfide (H 2 S)-synthesizing enzyme in cardiovascular cells. Overexpression of miR-499 by transfection of miR-499 mimics decreased CSE protein levels and intracellular H 2 S production in HCMVECs. Conclusions: Our results suggest that miR-499-induced CSE downregulation/inactivation thus H 2 S reduction may play an important role in diabetes-impaired angiogenic capacity. Silencing miR-499 in diabetic EPCs/ECs may provide a novel tool for the treatment of ischemic tissue injury in patients with diabetes.

TWEAK/Fn14 Signals Mediate Burn Wound Repair

TWEAK acts by engaging with Fn14 to regulate inflammatory responses, fibrosis, and tissue remodeling, which are central in the repair processes of wounds. This study aims to explore the potential role of the TWEAK/Fn14 pathway in the healing of cutaneous burn wounds. Third-degree burns were introduced in wild-type and Fn14-deficient BALB/c mice, followed by evaluation of wound areas and histological changes. The downstream cytokines including growth factors were also examined in lesional skin. Moreover, human dermal microvascular endothelial cells and dermal fibroblasts were analyzed invitro upon TWEAK stimulation. The healing of burn wounds was delayed in Fn14-deficient mice and was accompanied by the suppression of inflammatory responses, growth factor production, and extracellular matrix synthesis. Moreover, TWEAK/Fn14 activation enhanced the migration and cytokine production of both dermal microvascular endothelial cells and dermal fibroblasts. TWEAK also facilitates the expression of α-SMA and palladin in dermal fibroblasts. Furthermore, transfection of Fn14 small interfering RNA abrogated such promotion effect of TWEAK on these cells. In conclusion, TWEAK/Fn14 signals mediate the healing of burn wounds, possibly involving TWEAK regulation of the function of resident cells.

Abstract 330: Systemic Blocking Exosome Formation/Release Improves Ischemic Hindlimb Repair in Diabetic db/db Mice

Background: Recent studies demonstrated that plasma exosomes from diabetic patients and animals lose their ability to maintain physiological properties of cardiomyocytes. Exosome inhibitor GW 4869 (GW) tended to improve heart function in diabetic mice with myocardial infarction. We tested the hypotheses that systemic blocking exosome synthesis/formation improves ischemic hindlimb repair in diabetes. Methods and Results: Unilateral hindlimb ischemia (HLI) surgery was conducted by ligation of left femoral artery in 12-week male db/db and db/+ mice which were administrated with either GW (2 mg/kg body weight, i.p) or vehicle for 4 weeks starting from one week before HLI. Blood perfusion in ischemic hindlimbs was examined by Laser Doppler at pre-HLI, and 0, 3, 7, 14 and 21 days post-HLI. Plasma exosomes were isolated by standard ultracentrifugation method and counted by NanoSight. GW increased blood concentration of CD31 + /Sca-1 + cell 3-day post-HLI and decreased necrosis and loss of toe/toenail; improved blood flow; enhanced capillary/arterial density determined by CD31 and SMA-α staining and decreased fibrosis in the muscles of ischemic hindlimbs in db/db mice 21-day post-HLI. Plasma exosomes from db/db mice significantly impaired human cardiac microvascular endothelial cells (HCMVECs) tube formation and migration. Mechanistically, hepatocyte growth factor (HGF) expression was decreased in bone marrow-derived endothelial progenitor cells (EPCs) from db/db mice and HCMVECs treated with diabetic plasma exosomes. Administration of GW increased HGF level in diabetic EPCs. Furthermore, diabetic plasma exosomes decreased H3K4me3 (active maker of transcription) whereas increased H3K27me3 (suppressive marker of transcription) at HGF promoter. Finally, treatment of HCMEVs with exogenous HGF rescued diabetic exosomes impaired tube formation of HCMVEC. Conclusions: Systemic inhibition of exosome synthesis/formation by GW increased ischemic limb repair in diabetic db/db mice, at least partially, via histone methylation mediated suppression of HGF. Our findings suggest that therapeutic targeting of systemic dysfunctional exosomes could represent a new avenue for therapeutics of ischemic tissue injury in patients with diabetes.

Fasudil preserves lung endothelial function and reduces pulmonary vascular remodeling in a rat model of end‑stage pulmonary hypertension with left heart disease.

Pulmonary hypertension (PH) due to left heart disease (LHD) is a common condition associated with significant morbidity. It contributes to the elevation of pulmonary vascular resistance and mean pulmonary pressure, eventually leading to heart failure and even mortality. The present study aimed to explore the potential efficacy of late and long-term treatment with a Rho-kinase (ROCK) signaling inhibitor, namely fasudil, in a rat model of end-stage PH-LHD. The PH-LHD model was established by supracoronary aortic banding, and the effect of fasudil treatment on the progression of PH-LHD was monitored. After 9 weeks (63 days) of supracoronary aortic banding, a significant increase in mean pulmonary pressure and RV systolic pressure was observed in the rats, associated with increased RhoA/ROCK activity in the lungs. Therapy with fasudil (30 mg/kg/day, intraperitoneal) for 4 weeks from postoperative day 35 reversed the hemodynamic disorder and prevented pulmonary vascular remodeling in rats with PH-LHD. In addition, the blockade of ROCK signaling by fasudil decreased the protein levels of endothelin-1 (ET-1) and the mRNA expression levels of endothelin A receptor and promoted the production of nitric oxide (NO) in rats with PH-LHD. Furthermore, fasudil inhibited the migration of human pulmonary microvascular endothelial cells and the proliferation of pulmonary artery smooth muscle cells induced by ET-1. Therefore, this late, long-term blockade of the ROCK pathway by fasudil may be a promising strategy to reverse hemodynamic dysfunction and impede the development of end-stage PH-LHD in patients.

Native and recombinant phospholipases A2 of Scorpio maurus venom glands impair angiogenesis by targeting integrins α5β1 and αvβ3

We recently purified an heterodimeric phospholipase A2 named Sm-PLGV from the venom glands of scorpion Scorpio maurus containing a Long chain, a penta-peptide insertion, which is cut out during the maturation, followed by a short chain. Three recombinant forms of Sm-PLGV were produced in Escherichia coli: rPLA2(+5) containing the full-length sequence including the penta-peptide insert, rPLA2(−5) a fused continuous chain of the Long and the short chains without the penta-peptide and the Long chain alone without the short one. In this study, we showed that Sm-PLGV, rPLA2(+5) and rPLA2(−5) displayed more potent anti-angiogenic properties than the recombinant Long chain and the short chain obtained by chemical synthesis. These phospholipases A2 inhibited in a dose-dependent manner adhesion, migration and invasion of human microvascular endothelial cells through the alteration of α5β1 and αvβ3 integrins function. Using Matrigel™ and chick chorioallantoic membrane assays, we demonstrated that Sm-PLGV, rPLA2(+5) and rPLA2(−5) significantly inhibited both in vitro and in vivo angiogenesis. We also showed a clear dissociation of the anti-angiogenic effect of Sm-PLGV and its catalytic activity. This is the first study describing an anti-angiogenic effect for recombinant scorpion venom enzymes.

17β-Estradiol Promotes Angiogenesis of Rat Cardiac Microvascular Endothelial Cells In Vitro.

BackgroundThe formation of new blood vessels, known as angiogenesis, is critical for recovery from ischemic heart disease, and estrogen is considered an important factor in this process. Here, we investigated the effects of 17β-estradiol (17β-E2) on proliferation and migration of cardiac microvascular endothelial cells (CMECs) in vitro.Material/MethodsRat CMECs were isolated and cultured with 17β-E2 (0.001–1 μmol/l) in the absence or presence of the estrogen antagonist tamoxifen. Then, the expression level of estrogen receptor alpha was evaluated by using immunofluorescence assay, RT-PCR, and Western blot. Cell proliferation was detected by methyl thiazolyl tetrazolium analysis and the cell migration was verified by a scraping assay and quantified by a Transwell chamber assay. CMEC differentiation was examined using a tube formation assay. Vascular endothelial growth factor (VEGF) secretion was detected by enzyme-linked immunosorbent assay.ResultsCMECs exhibited homogenous, polygonal, exhibited contact inhibition, and had characteristically ovoid nuclei with 1 or 2 nucleoli, and the cytoplasm exhibited red fluorescence after staining for von Willebrand factor. 17β-E2 treatment upregulated estrogen receptor alpha expression in CMECs. 17β-E2 treatment significantly promoted the proliferation, migration, tubular structure formation, and VEGF secretion in CMECs. The maximal proliferation occurred in the presence of 0.01 μmol/l 17β-E2. Furthermore, estrogen and VEGF were found to synergistically stimulate angiogenesis.ConclusionsOur data show that 17β-E2 promotes angiogenesis in vitro and suggests that estrogen treatment as a novel therapeutic modality in the management of arterial insufficiency.

WITHDRAWN: Galectin-3 carbohydrate recognition domain induces microvascular endothelial cell migration by activating β1 Integrin

// Zhongyu Zhang 1 , Yuan Guan 1 , Xiyao Yu 1 , Hairong Cheng 1 , Yifa Zhou 1 and Guihua Tai 1 1 School of Life Sciences, Northeast Normal University, Changchun, PR China Correspondence to: Guihua Tai, email: taigh477@nenu.edu.cn Keywords : galectin-3; CRD; migration; HMEC-1; β1 integrin Received: August 18, 2017 Accepted: January 02, 2018 Epub: January 11, 2018 Abstract Galectin-3 (Gal-3) is composed of a long N-terminal tail (NT) connected to its conserved carbohydrate recognition domain (CRD). The NT is degraded by matrix metalloproteinases and prostate-specific antigen. Here, we demonstrate that NT-truncated Gal-3 variants with deletion of the first 12 or 68 residues, or the entire 110-residues NT, triggers migration of human microvascular endothelial cells (HMEC- 1). Using mass spectrometry and pull-down analysis, we show that the Gal-3 CRD binds to and activates cell surface β1 integrin promoting phosphorylation of FAK. Antibody blocking assays suggested that α2, α4, and α6 integrins are involved, with α2 and β1 forming a heterodimer. shRNA knockdown or antibody-mediated inhibition of β1 integrin function inhibited CRD-induced cell migration and attenuated FAK phosphorylation. FAK-specific inhibitors also inhibited Gal-3 CRD-induced cell migration. Interestingly, full-length Gal-3 and its CRD promoted similar effects. These findings highlight the need to reconsider Gal-3 NT cleavage in formulating strategies to address tumor angiogenesis.

Efficacy of Lenvatinib, a multitargeted tyrosine kinase inhibitor, on laser-induced CNV mouse model of neovascular AMD

Neovascular age-related macular degeneration (AMD) is a leading cause of vision loss worldwide. Although intravitreal injection of anti-VEGF antibodies and VEGF Trap have significant clinical benefits, the complications of intravitreal injection, drug resistance and patient compliance still need to be concerned. In this study, the effects of an orally administered multi-targeted tyrosine kinase inhibitor (Lenvatinib, E7080) were evaluated in vitro and in vivo on neovascular AMD mouse model. The results showed that E7080 effectively inhibited the proliferation, migration and tubule formation of human choroidal microvascular endothelial cells (HCMECs), and suppressed the angiogenesis of zebrafish subintestinal vessels without causing malformation. The anti-angiogenic effect of E7080 on the laser-induced choroidal neovascularization (CNV) mouse model by oral administration of 10 mg/kg/day was observed. The fluorescein angiography showed CNV leakage area in treatment group vs control group was 3.407 ± 0.2939 vs 5.202 ± 0.9001 (P = .0133) at day 7th post laser-induced CNV, 1.138 ± 0.4334 vs 3.122 ± 0.3466 (P = .0064) at day 14th, 1.401 ± 0.6577 vs 2.781 ± 0.9815 (P = .00262) at day 21th respectively. Moreover, pharmacokinetics analysis in rat retina showed that E7080 rapidly penetrated the blood-retina barrier to retina through oral administration. The T1/2 in retina was 3.81 ± 0.77 h, the Tmax was 4.60 ± 0.73 h, the AUC0-∞ was 110448.51 ± 18532.51 h*ng/g after a single dose administration analyzed by liquid chromatography-tandem mass spectrometry (LC/MS/MS). In conclusion, our study suggested that orally administered E7080 can be a novel therapeutic strategy for neovascular AMD.

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Neovascular age-related macular degeneration (AMD) is a leading cause of vision loss worldwide. Although intravitreal injection of anti-VEGF antibodies and VEGF Trap have significant clinical benefits, the complications of intravitreal injection, drug resistance and patient compliance still need to be concerned. In this study, the effects of an orally administered multi-targeted tyrosine kinase inhibitor (Lenvatinib, E7080) were evaluated in vitro and in vivo on neovascular AMD mouse model. The results showed that E7080 effectively inhibited the proliferation, migration and tubule formation of human choroidal microvascular endothelial cells (HCMECs), and suppressed the angiogenesis of zebrafish subintestinal vessels without causing malformation. The anti-angiogenic effect of E7080 on the laser-induced choroidal neovascularization (CNV) mouse model by oral administration of 10 mg/kg/day was observed. The fluorescein angiography showed CNV leakage area in treatment group vs control group was 3.407 ± 0.2939 vs 5.202 ± 0.9001 (P = .0133) at day 7th post laser-induced CNV, 1.138 ± 0.4334 vs 3.122 ± 0.3466 (P = .0064) at day 14th, 1.401 ± 0.6577 vs 2.781 ± 0.9815 (P = .00262) at day 21th respectively. Moreover, pharmacokinetics analysis in rat retina showed that E7080 rapidly penetrated the blood-retina barrier to retina through oral administration. The T1/2 in retina was 3.81 ± 0.77 h, the Tmax was 4.60 ± 0.73 h, the AUC0-∞ was 110448.51 ± 18532.51 h*ng/g after a single dose administration analyzed by liquid chromatography-tandem mass spectrometry (LC/MS/MS). In conclusion, our study suggested that orally administered E7080 can be a novel therapeutic strategy for neovascular AMD.

Claudin-5 regulates blood-brain barrier permeability by modifying brain microvascular endothelial cell proliferation, migration, and adhesion to prevent lung cancer metastasis.

To investigate the roles of Claudin-5 (CLDN5) in regulating the permeability of the blood-brain barrier (BBB) during lung cancer brain metastasis. By silencing and overexpressing the CLDN5 gene in human brain vascular endothelial (hCMEC/D3) cells, we demonstrated the attenuation of cell migration ability and CLDN5's significant positive role in cell proliferation in CLDN5-overexpressing hCMEC/D3 cells and observed the opposite result in the CLDN5 knockdown group. The reinforced CLDN5 expression reduced the paracellular permeability of hCMEC/D3 cells and decreased the invasion of lung adenocarcinoma A549 cells. Overall, 1685 genes were found to be differentially expressed between the CLDN5-overexpressing cells and the control cells using the Affymetrix Human Transcriptome Array 2.0 (HTA 2.0), and the function of these genes was determined by Gene Ontology and pathway analyses. The possible biological functions of the 1685 genes include cell proliferation, adhesion molecules, and the Jak-STAT, PI3K-Akt, Wnt, and Notch signaling pathways. The identified sets of mRNAs that were specific to CLDN5-overexpressing hCMEC/D3 cells were verified by a qRT-PCR experiment. CLDN5 regulates the permeability of BBB by regulating the proliferation, migration, and permeability of hCMEC/D3 cells, especially through the cell adhesion molecule signaling pathway, to enhance the function of the tight junctions, which was involved in reducing the formation of lung cancer brain metastasis.

Relative distribution and biological characterization of CXCL4L1 isoforms in platelets from healthy donors

CXCL4L1, a platelet-derived ELR-negative CXC chemokine, is a powerful angiostatic and anti-tumoral chemokine. We developed a mass spectrometric assay for the detection of different natural CXCL4L1 isoforms. Using this assay, we identified 4 different CXCL4L1 isoforms in the supernatant of thrombin-stimulated platelets from healthy volunteers: the classical isoform CXCL4L1(1-70), CXCL4L1(-4-70), which probably arises through alternative signal peptide removal and two COOH-terminally truncated isoforms CXCL4L1(1-69) and CXCL4L1(-4-69). CXCL4L1(1-70) was the most abundant isoform, whereas CXCL4L1(-4-70) was detected in 50% of the platelet preparations. Since alterations to the NH2-terminus of chemokines can have severe biological consequences, we investigated the impact of the extension with 4 NH2-terminal amino acids on the biological activity of CXCL4L1. In vitro, CXCL4L1(-4-70) was as potent as CXCL4L1(1-70) in inhibiting signal transduction and migration of human microvascular endothelial cells towards vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2). In a FITC-conjugated dextran cell permeability assay, both splice variants showed a strong but comparable anti-permeable effect upon VEGF stimulation of the endothelial cell monolayer. In vivo angiogenesis induced by FGF-2 was equally reduced by CXCL4L1(1-70) and CXCL4L1(-4-70). In chemotaxis assays with CXCR3A-transfected cells the CXCL4L1 isoforms both induced migration from 125ng/ml onward. Finally, CXCL4L1(1-70) and CXCL4L1(-4-70) showed the same affinity for heparin. In conclusion, the investigated biological activities of CXCL4L1 are not influenced by the four extra NH2-terminal residues present in the alternatively spliced isoform CXCL4L1(-4-70). Therefore, our results suggest that both isoforms equally interact with the CXCR3A and CXCR3B receptor.

The impact of transcription factor Fli1 deficiency on the regulation of angiogenesis.

The insufficiency of Friend leukaemia virus integration 1 (Fli1), a member of the Ets family transcription factors, is implicated in the pathogenesis of vasculopathy associated with systemic sclerosis (SSc). Fli1 deficiency accelerates early steps of angiogenesis, including detachment of pre-existing pericytes and extracellular matrix degradation by endothelial proteinases, but the impact of Fli1 deficiency on the other steps of angiogenesis has not been investigated. Therefore, we evaluated the effect of Fli1 deficiency on migration, proliferation, cell survival and tube formation of human dermal microvascular endothelial cells (HDMECs). HDMECs transfected with FLI1 siRNA exhibited a greater migratory property in scratch assay and transwell migration assay and a higher proliferation rate in BrdU assay than HDMECs transfected with non-silencing scrambled RNA. In flow cytometry-based apoptosis assay, FLI1 siRNA-transduced HDMECs revealed the decreased number of annexin and propidium iodide-double-positive apoptotic cells compared with control cells, reflecting the promotion of cell survival. On the other hand, tubulogenic activity on Matrigel was remarkably suppressed in Fli1-deficient HDMECs relative to control cells. These results indicate that Fli1 deficiency promotes migration, proliferation and cell survival, while abating tube formation of endothelial cells, suggesting that Fli1 deficiency is potentially attributable to the development of both proliferative obliterative vasculopathy (occlusion of arterioles and small arteries) and destructive vasculopathy (loss of small vessels) characteristic of SSc vasculopathy.