Proliferation Of Microvascular Endothelial Cells Research Articles

Ginsenoside F1 promotes angiogenesis by activating the IGF-1/IGF1R pathway

Ischemic stroke is one of the most lethal and highly disabling diseases that seriously affects the human health and quality of life. A therapeutic angiogenic strategy has been proposed to alleviate ischemia-induced injury by promoting angiogenesis and improving cerebrovascular function in the ischemic regions. The insulin-like growth factor 1 (IGF-1)/insulin-like growth factor 1 receptor (IGF1R) axis is crucial for cerebral angiogenesis and neurogenesis. However, effective drugs that prevent cerebral ischemic injury by inducing cerebral angiogenesis via activation of the IGF1R pathway are lacking. Here, we screened a pro-angiogenic agent ginsenoside F1 (GF1), a ginseng saponin isolated from a traditional Chinese medicine that was widely used in ischemic stroke treatment. It promoted the proliferation, mobility and tube formation of human umbilical vein endothelial cells and human brain microvascular endothelial cells, as well as pericytes recruitment to the endothelial tubes. GF1 stimulated vessel sprouting in the rat arterial ring and facilitated neovascularization in chicken embryo chorioallantoic membrane (CAM). In the in vivo experiments, GF1 rescued the axitinib-induced vascular defect in zebrafish. It also increased the microvessel density (MVD) and improved focal cerebral blood perfusion in the rat middle cerebral artery occlusion (MCAO) model. Mechanism studies revealed that GF1-induced angiogenesis depended on IGF1R activation mediated by the autocrine IGF-1 loop in endothelial cells. Based on our findings, GF1-induced activation of the IGF-1/IGF1R pathway to promote angiogenesis is an effective approach to alleviate cerebral ischemia, and GF1 is a potential agent that improves cerebrovascular function and promotes recovery from ischemic stroke.

Isoform‐specific Pde3 knockout mice spontaneously develop pulmonary hypertension

Pulmonary hypertension (PH) is associated with vasoconstriction and vascular remodeling characterized by pulmonary artery smooth muscle cell (PASMC) and microvascular endothelial cell (PMVEC) proliferation and dysfunction. Phosphodiesterase 3 (PDE3), of which there are two isoforms (3A and 3B), hydrolyzes cAMP and cGMP, both critical cyclic nucleotides in cellular function. While both isoforms are present in the pulmonary vasculature, their specific contributions to the regulation of pulmonary vascular tone are unclear. Non‐selective PDE3 inhibitors have been used to treat PH for their vasodilatory effect, presumably by increasing cellular cAMP levels. We hypothesize that isoform‐selective knockout of PDE3 may reveal distinct roles in the regulation of pulmonary vascular tone and/or PH pathogenesis. Adult (8–10 weeks) Pde3a−/− (3A‐KO), Pde3b−/− (3B‐KO), and their respective wild type (WT) mice (3A‐WT, 3B‐WT) were exposed to normoxia or normobaric hypoxia (10% O2) for 28 days. Consistent with previous reports, we found that 3A‐WT and 3B‐WT mice exposed to 10% O2 for 28 days had right ventricular (RV) hypertrophy as evidenced by an increased RV/[left ventricle (LV)+septum (S)] weight ratio relative to mice exposed to 21% O2 [p < 0.001; 0.38 ± 0.01 vs 0.25 ± 0.01 (3A) and 0.35 ± 0.01 vs 0.24 ± 0.01 (3B)]. In hypoxia, 3A‐WT and 3B‐WT mice had elevated pulmonary artery pressures estimated by 2D echocardiography measurements of pulmonary acceleration time/ejection time (PAT/PET) (p < 0.0001; 0.21 ± 0.004 vs 0.29 ± 0.01 (3A) and 0.21 ± 0.01 vs 0.31 ± 0.01 (3B) relative to mice exposed to 21% O2. Interestingly, in normoxia both 3A‐KO (p = 0.012; 0.25 ± 0.004 vs 0.29 ± 0.01 and 3B‐KO (p = 0.023; 0.26 ± 0.01 vs 0.31 ± 0.01) mice had evidence of elevated pulmonary artery pressures relative to their respective WT mice, as evidenced by a lower PAT/PET via echocardiography. Consistent with these results, in normoxia RV/(LV+S) was higher in both 3A‐KO (0.29 ± 0.01 vs 0.25 ± 0.01, p = 0.0008) and 3B‐KO (0.31 ± 0.01 vs 0.24 ± 0.01, p < 0.0001) than in their respective normoxiaexposed WT. Normoxia‐exposed 3B‐KO mice also had elevated right ventricular systolic pressure compared to normoxia‐exposed 3B‐WT (28.9 ± 0.7 mmHg vs 24.2 ± 1.2 mmHg, p = 0.0028). In hypoxia, there was no difference in RV/(LV+S) between 3A‐WT and 3A‐KO mice, although lower estimated pulmonary artery pressures were seen in the 3A‐KO mice (p = 0.0043). Conversely, in hypoxia there was a trend towards higher RV/(LV+S) in the 3B‐KO compared to 3B‐WT mice (p = 0.0532; 0.42 ± 0.02 vs 0.35 vs ± 0.01), but no difference was seen in pulmonary artery pressures between hypoxia‐exposed, 3B‐KO and 3B‐WT mice. Our data suggest a complex and isoform‐specific role of the Pde3 genes in the pathogenesis of PH associated with hypoxia. Furthermore, these data suggest a key role for each of the PDE3 isoforms in the maintenance of the low pulmonary vascular resistance found in normal lungs.Support or Funding Information5K08HL105677‐01A1, R01HL136963‐01A1This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.

Inhibition of mitochondrial autophagy protects donor lungs for lung transplantation against ischaemia-reperfusion injury in rats via the mTOR pathway.

Impaired mitochondrial function is a key factor attributing to lung ischaemia‐reperfusion (IR) injury, which contributes to major post‐transplant complications. Thus, the current study was performed to investigate the role of mitochondrial autophagy in lung I/R injury and the involvement of the mTOR pathway. We established rat models of orthotopic left lung transplantation to investigate the role of mitochondrial autophagy in I/R injury following lung transplantation. Next, we treated the donor lungs with 3‐MA and Rapamycin to evaluate mitochondrial autophagy, lung function and cell apoptosis with different time intervals of cold ischaemia preservation and reperfusion. In addition, mitochondrial autophagy, and cell proliferation and apoptosis of pulmonary microvascular endothelial cells (PMVECs) exposed to hypoxia‐reoxygenation (H/R) were monitored after 3‐MA administration or Rapamycin treatment. The cell apoptosis could be inhibited by mitochondrial autophagy at the beginning of lung ischaemia, but was rendered out of control when mitochondrial autophagy reached normal levels. After I/R of donor lung, the mitochondrial autophagy was increased until 6 hours after reperfusion and then gradually decreased. The elevation of mitochondrial autophagy was accompanied by promoted apoptosis, aggravated lung injury and deteriorated lung function. Moreover, the suppression of mitochondrial autophagy by 3‐MA inhibited cell apoptosis of donor lung to alleviate I/R‐induced lung injury as well as inhibited H/R‐induced PMVEC apoptosis, and enhanced its proliferation. Finally, mTOR pathway participated in I/R‐ and H/R‐mediated mitochondrial autophagy in regulation of cell apoptosis. Inhibition of I/R‐induced mitochondrial autophagy alleviated lung injury via the mTOR pathway, suggesting a potential therapeutic strategy for lung I/R injury.

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.

Secretogranin III as a novel target for the therapy of choroidal neovascularization

Wet age-related macular degeneration (AMD) with choroidal neovascularization (CNV) is a leading cause of vision loss in the elderly. The advent of anti-vascular endothelial growth factor (VEGF) drugs represents a major breakthrough in wet AMD therapy but with limited efficacy to improve visual acuity. Secretogranin III (Scg3, SgIII) was recently discovered as a novel angiogenic factor with VEGF-independent mechanisms. Scg3-neutralizing monoclonal antibody (mAb) was reported to alleviate pathological retinal neovascularization in oxygen-induced retinopathy mice and retinal vascular leakage in diabetic mice with high efficacy and disease selectivity. Herein we investigated whether Scg3 is a novel angiogenic target for CNV therapy in mouse models. We found that anti-Scg3 ML49.3 mAb inhibited Scg3-induced proliferation and Src phosphorylation in human retinal microvascular endothelial cells. Intravitreal injection of Scg3-neutralizing polyclonal antibodies (pAb) or mAb significantly attenuated laser-induced CNV leakage, CNV 3D volume, lesion area and vessel density. Furthermore, subcutaneous administration of Scg3-neutralizing pAb or mAb significantly prevented Matrigel-induced CNV. The efficacy of anti-Scg3 pAb or mAb was comparable to VEGF inhibitor aflibercept. These findings suggest that Scg3 plays an important role in CNV pathogenesis and that anti-Scg3 mAb efficiently ameliorates laser- or Matrigel-induced CNV.

Advanced glycation end products facilitate the proliferation and reduce early apoptosis of cardiac microvascular endothelial cells via PKCβ signaling pathway: Insight from diabetic cardiomyopathy.

Objective:To investigate the effects of advanced glycation end products (AGEs) on the proliferation and apoptosis of cardiac microvascular endothelial cells (CMECs) in rats and their underlying signaling pathway.Methods:CMECs were isolated from Sprague–Dawley rats. We first examined the effects of AGEs on the proliferation and apoptosis of CMECs and then tested whether protein kinase C (PKC) β blockers could counteract the effects of AGEs. The PKC agonists phorbol 12-myristate 13-acetate (PMA) and PKCβ blockers were also used to verify whether PKC could act independently on CMECs. The receptor for AGEs (RAGE)–small interfering RNA (siRNA) transfection was used to verify the effect of AGEs on PKC. Following the above steps, we explained whether AGEs regulated the CMEC proliferation and early apoptosis through the PKCβ signaling pathway. Proliferation of CMECs was detected using the Cell Counting Kit-8 (CCK-8) assay, and early apoptosis was determined using the Annexin V- Fluorescein Isothiocyanate (FITC)/propidium iodide (PI) double staining. Expression of proliferation and apoptosis-related proteins and PKC phosphorylation were determined by western blotting analysis. Cell cycle distributions were assayed using a BD FACSCalibur cell-sorting system.Results:AGEs facilitated the proliferation of CMECs, upregulated phosphorylated extracellular signal regulated kinase (p-ERK), and accelerated the entry of cells from G1 phase to the S+G2/M phase, which was consistent with the upregulated cyclin D1 by AGEs. AGEs inhibited early apoptosis of CMECs by increasing the expression of survivin and decreasing the expression of cleaved-caspase3. All these effects can be reversed by PKCβ1/2inhibitors. In addition, AGE upregulated the RAGE expression and phosphorylation of PKCβ1/2 in CMECs, while the inhibition of RAGE reversed the phosphorylation, as well as the effects of AGEs on proliferation and apoptosis in CMECs.Conclusion:The study indicated that AGEs facilitated the proliferation and reduced early apoptosis of CMECs via the PKCβ signaling pathway.

MiR145-5p inhibits proliferation of PMVECs via PAI-1 in experimental hepatopulmonary syndrome rat pulmonary microvascular hyperplasia.

ABSTRACTHepatopulmonary syndrome (HPS) is a triad of advanced liver disease, intrapulmonary vasodilatation and arterial hypoxemia. Increasing evidence shows that HPS is associated with pulmonary microvascular hyperplasia. The aim of this work was to investigate the underlying mechanism of miR-145 in regulating the proliferation of pulmonary microvascular endothelial cells (PMVECs) and angiogenesis in HPS via plasminogen activator inhibitor-1 (PAI-1). To test this, morphology score and number of pulmonary microvascular were assessed in lung tissues from rats with HPS by Hematoxylin and Eosin (H&E) staining. Expression levels of PAI-1 were assessed in lung tissues from HPS rats, as well as in PMVECs treated with HPS rat serum. We also selected the putative microRNA binding site on PAI-1 by bioinformatics analysis. Then, miR145-3p and miR145-5p expression levels in the lungs and PMVECs of rats were detected by qRT-PCR because miR145-5p is a microRNA binding site on PAI-1. In addition, the effects of miR-145-5p regulation on PAI-1 were examined by upregulation and downregulation of miR-145-5p and specific lentivirus transfection was used to overexpress and knockdown PAI-1 to assess PAI-1 function on PMVECs proliferation. Our data showed that levels of PAI-1 expression in lung tissue of rats increased significantly when rats were treated with common bile duct ligation. We found that levels of miR-145-5p were frequently downregulated in HPS tissues and cell lines, and overexpression of miR-145-5p dramatically inhibited PMVECs proliferation. We further verified PAI-1 as a novel and direct target of miR-145-5p in HPS. MiR-145-5p inhibits PAI-1 synthesis and the expression changes of PAI-1 directly affect the proliferation of PMVECs. We concluded that miR-145-5p negatively regulates PMVEC proliferation through PAI-1 expression. In addition, overexpression of miR-145-5p may prove beneficial as a therapeutic strategy for HPS treatment.

Effect of natural hirudin on angiogenesis of human microvascular endothelial cells

To explore the effect of natural hirudin on proliferation of human microvascular endothelial cells (HMVECs) and its preliminary mechanism of promoting angiogenesis. Three-dimensional culture models of HMVECs were established in vitro and observed by inverted phase contrast microscopy after 24 hours of culturing. Then, the three-dimensional culture models of HMVECs were treated with different concentrations (1, 4, and 7 ATU/mL) of the natural hirudin, respectively, and Dulbecco's modified Eagle's medium containing 10% fetal bovine serum as control. The cell proliferations of 4 groups were detected by cell counting kit 8 (CCK-8) method at 24, 48, and 72 hours; the angiogenesis of 4 groups were observed by tube formation assay at 24 hours; the expressions of vascular endothelial growth factor (VEGF) and Notch1 of HMVECs in 4 groups were observed by immunofluorescence staining at 24 hours. The observation of cells in three-dimensional culture models showed that HMVECs attached to Matrigel well, and the cells formed tube structure completely after 24 hours. The results of CCK-8 test showed that the absorbance ( A) value of 1 and 4 ATU/mL groups were higher than that of control group at each time point ( P<0.05), and A value of 4 ATU/mL group was the highest. The A value of 7 ATU/mL group was significantly lower than those of 1 and 4 ATU/mL groups and control group ( P<0.05). The tube formation assay showed that the tube structure was more in 1 and 4 ATU/mL groups than in 7 ATU/mL group and control group, and in 4 ATU/mL group than in 1 ATU/mL group, showing significant differences ( P<0.05). There was no significant difference between 7 ATU/mL group and control group ( P>0.05). The results of immunofluorescence staining showed that compared with control group, the Notch1 expression was higher in 1 and 4 ATU/mL groups and lower in 7 ATU/mL group; and there was significant difference between 4 and 7 ATU/mL groups and control group ( P<0.05). The VEGF expression was higher in 1, 4, and 7 ATU/mL groups than in control group, in 4 ATU/mL group than in 1 and 7 ATU/mL groups, showing significant differences ( P<0.05). Natural hirudin can promote angiogenesis at low and medium concentrations, but suppress angiogenesis at high concentrations. Its mechanism may be related to the VEGF-Notch signal pathway.

Design and synthesis of a native heparin disaccharide grafted poly‑2‑aminoethyl methacrylate glycopolymer for inhibition of melanoma cell metastasis

Numerous studies have proved that heparin, a sub-group of glycosaminoglycan, possesses great potential as anti-metastasis agent. However, the native strong anti-coagulant activity which causes serious side effects, such as bleeding, has limited its clinical applications for safety concern. To overcome this problem, we synthesized a panel of novel glycopolymers that mimic heparin structure with substantially reduced anti-coagulant activity by a simple grafted-on strategy. The influence of molecular weight & distribution, substituting degree and sulfonic density on cytotoxicity were determined by systematic MTT analysis to select the candidates with highly bio-compatibility. Among these glycopolymers, a sulfated poly‑2‑aminoethyl methacrylate grafted with heparin disaccharide (abbreviated as SGPHD) has shown potent efficacy in inhibition of heparanase activity and microvascular endothelial cell proliferation in vitro. Further experiments demonstrated that SGPHD inhibited B16 murine melanoma cell migration, invasion and adhesion to platelets or microvascular endothelial cells, thus, presented as a possible anti-metastatic agent by providing a whole course protection during tumor metastasis. The results will have significant impacts for the further rational design of glycopolymer medicine.

X-box binding protein l splicing attenuates brain microvascular endothelial cell damage induced by oxygen-glucose deprivation through the activation of phosphoinositide 3-kinase/protein kinase B, extracellular signal-regulated kinases, and hypoxia-inducible factor-1α/vascular endothelial growth factor signaling pathways.

Angiogenesis is positively correlated with the survival rate of stroke patients. Therefore, studying factors that initiate and promote angiogenesis after ischemic stroke is crucial for finding novel and effective treatment targets that improve the prognosis of stroke. X-box binding protein l splicing (XBP1s) plays a positive regulatory role in cell proliferation and angiogenesis. However, the role and mechanism of XBP1s on the proliferation of brain microvascular endothelial cells (BMECs) and angiogenesis after cerebral ischemia remains unclear. In the current study, we investigated the role XBP1s plays in BMEC proliferation and angiogenesis following cerebral ischemia. In this study, the roles of XBP1s on cell survival, apoptosis, cycle migration, and angiogenesis were determined in oxygen-glucose deprivation (OGD) treated BMECs. The expression of XBP1s in BMECs, which were exposed to OGD at 0, 2, 4, and 6 hr, increased in a time-dependent manner. The overexpression of XBP1s promoted cell survival, cell cycle, migration, and angiogenesis of BMECs, and inhibited the apoptosis in OGD-treated BMECs. In addition, the overexpression of XBP1s promoted the expression of cyclin D1, matrix metalloproteinase (MMP-2), and MMP-9, but inhibited cleaved Caspase-3 and cleaved Caspase-9 expression in OGD-treated BMECs. The overexpression of XBP1s also promoted the expression of hypoxia-inducible factor 1-alpha, vascular endothelial growth factor, phosphatidylinositol-4,5-bisphosphate 3-kinase, p-AKT, p-mTOR, p-GSK3β, and p-extracellular signal-regulated kinase1/2 in OGD-treated BMECs. The effect of XBP1s silencing was opposite to that of XBP1s overexpression. In conclusion, using an in vitro OGD model, we demonstrated that XBP1s may be a promising target for ischemic stroke therapy to maintain BMECs survival and induce angiogenesis.

GW29-e0226 Study on the Mechanism of Mobilization EPCs in Myocardial Infarction Mice by Naoxintong Capsule

Naoxintong Capsule is classical Chinese medicine to treat with stroke and cardiovascular disease.And it has been reported to increase the proliferation of microvascular endothelial cells in the normal situations.In this study,we investigate that Naoxintong Capsule whether can mobilization marrow

Small Molecule Derived From Carboxyethylpyrrole Protein Adducts Promotes Angiogenesis in a Mouse Model of Peripheral Arterial Disease.

BackgroundCEP (ω‐[2‐carboxyethyl]pyrrole) protein adducts are the end products of lipid oxidation associated with inflammation and have been implicated in the induction of angiogenesis in pathological conditions such as tissue ischemia. We synthesized small molecules derived from CEP protein adducts and evaluated the angiogenic effect of the CEP analog CEP03 in the setting of peripheral arterial disease.Methods and ResultsThe angiogenic effect of CEP03 was assessed by in vitro analysis of primary human microvascular endothelial cell proliferation and tubelike formation in Matrigel (Corning). In the presence of CEP03, proliferation of endothelial cells in vitro increased by 27±18% under hypoxic (1% O2) conditions, reaching similar levels to that of VEGFA (vascular endothelial growth factor A) stimulation (22±10%), relative to the vehicle control treatment. A similar effect of CEP03 was demonstrated in the increased number of tubelike branches in Matrigel, reaching >70% induction in hypoxia, compared with the vehicle control. The therapeutic potential of CEP03 was further evaluated in a mouse model of peripheral arterial disease by quantification of blood perfusion recovery and capillary density. In the ischemic hind limb, treatment of CEP03 encapsulated within Matrigel significantly enhanced blood perfusion by 2‐fold after 14 days compared with those treated with Matrigel alone. Moreover, these results concurred with histological finding that treatment of CEP03 in Matrigel resulted in a significant increase in microvessel density compared with Matrigel alone.ConclusionsOur data suggest that CEP03 has a profound positive effect on angiogenesis and neovessel formation and thus has therapeutic potential for treatment of peripheral arterial disease.

Stimulation of angiogenesis using single-pulse low-pressure shock wave treatment.

Endothelial cells respond to mechanical stimuli such as stretch. This property can be exploited with caution to induce angiogenesis which will have immense potential to treat pathological conditions associated with insufficient angiogenesis. The primary aim of this study is to test if low-pressure shock waves can be used to induce angiogenesis. Using a simple diaphragm-based shock tube, we demonstrate that a single pulse of low pressure (0.4bar) shock wave is enough to induce proliferation in bovine aortic endothelial cells and human pulmonary microvascular endothelial cells. We show that this is associated with enhanced Ca++ influx and phosphorylation of phosphatidylinositol-3-kinase (PI3K) which is normally observed when endothelial cells are exposed to stretch. We also demonstrate the pro-angiogenic effect of shock waves of single pulse (per dose) using murine back punch wound model. Shock wave treated mice showed enhanced wound-induced angiogenesis as reflected by increased vascular area and vessel length. They also showed accelerated wound closure compared to control mice. Overall, our study shows that just a single pulse/shot (per dose) of shock waves can be used to induce angiogenesis. Importantly, we demonstrate this effect using a pulse of low-pressure shock waves (0.4bar, in vitro and 0.15bar, in vivo). KEY MESSAGES: Low-pressure single-pulse shock waves can induce endothelial cell migration and proliferation. This effect is endothelial cell specific. These shock waves enhance wound-induced angiogenesis in vivo. These shock waves can also accelerate wound healing in vivo.

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.

Inhibition of miR-21-5p suppresses high glucose-induced proliferation and angiogenesis of human retinal microvascular endothelial cells by the regulation of AKT and ERK pathways via maspin.

The aim of the present study is to investigate the role of miR-21-5p in angiogenesis of human retinal microvascular endothelial cells (HRMECs). HRMECs were incubated with 5mM glucose, 30mM glucose or 30mM mannitol for 24h, 48h or 72h. Then, HRMECs exposed to 30mM glucose were transfected with miR-21-5p inhibitor. We found that high glucose increased the expression of miR-21-5p, VEGF, VEGFR2 and cell proliferation activity. Inhibition of miR-21-5p reduced high glucose-induced proliferation, migration, tube formation of HRMECs, and reversed the decreased expression of maspin as well as the abnormal activation of PI3K/AKT and ERK pathways. Down-regulation of maspin by siRNA significantly increased the activities of PI3K/AKT and ERK pathways. In conclusion, inhibition of miR-21-5p could suppress high glucose-induced proliferation and angiogenesis of HRMECs, and these effects may partly dependent on the regulation of PI3K/AKT and ERK pathways via its target protein maspin.

Fn14 deficiency ameliorates psoriasis-like skin disease in a murine model

Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) is a multifunctional cytokine that acts through its receptor fibroblast growth factor-inducible 14 (Fn14). Recent studies demonstrated that the TWEAK/Fn14 signals participate in the development of psoriasis. The purpose of this study was to further explore the effect of Fn14 inhibition on experimental psoriasis. Psoriasis-like skin disease was induced in the wild-type and Fn14-knockout BALB/c mice. We found that Fn14 deficiency ameliorates psoriasis-like lesion in this model, accompanied by less inflammatory cell infiltration and proinflammatory cytokine production in lesional skin. The cutaneous expression of TNF receptor type 2 also decreased in the Fn14-deficient mice. Moreover, the topical application of TWEAK exacerbated psoriatic lesion in the wild-type but not in the Fn14-deficient mice. Furthermore, TWEAK promoted the expression of interleukin 8, keratin 17, and epidermal growth factor receptor (EGFR) but inhibited the expression of involucrin in psoriatic keratinocytes in vitro. Interestingly, such effect of TWEAK was abrogated by an EGFR inhibitor (erlotinib). TWEAK also enhances the proliferation and interleukin-6 production of dermal microvascular endothelial cells under psoriatic condition. In conclusion, TWEAK/Fn14 signals contribute to the development of psoriasis, and involves the modulation of resident cells and the transduction of the EGFR pathway. Fn14 inhibition might be a novel therapeutic strategy for patients with psoriasis.

Nintedanib improves cardiac fibrosis but leaves pulmonary vascular remodelling unaltered in experimental pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is associated with increased levels of circulating growth factors and corresponding receptors such as platelet derived growth factor, fibroblast growth factor and vascular endothelial growth factor. Nintedanib, a tyrosine kinase inhibitor targeting primarily these receptors, is approved for the treatment of patients with idiopathic pulmonary fibrosis. Our objective was to examine the effect of nintedanib on proliferation of human pulmonary microvascular endothelial cells (MVEC) and assess its effects in rats with advanced experimental pulmonary hypertension (PH). Proliferation was assessed in control and PAH MVEC exposed to nintedanib. PH was induced in rats by subcutaneous injection of Sugen (SU5416) and subsequent exposure to 10% hypoxia for 4 weeks (SuHx model). Four weeks after re-exposure to normoxia, nintedanib was administered once daily for 3 weeks. Effects of the treatment were assessed with echocardiography, right heart catheterization, and histological analysis of the heart and lungs. Changes in extracellular matrix production was assessed in human cardiac fibroblasts stimulated with nintedanib. Decreased proliferation with nintedanib was observed in control MVEC, but not in PAH patient derived MVEC. Nintedanib treatment did not affect right ventricular (RV) systolic pressure or total pulmonary resistance index in SuHx rats and had no effects on pulmonary vascular remodelling. However, despite unaltered pressure overload, the right ventricle showed less dilatation and decreased fibrosis, hypertrophy, and collagen type III with nintedanib treatment. This could be explained by less fibronectin production by cardiac fibroblasts exposed to nintedanib. Nintedanib inhibits proliferation of pulmonary MVECs from controls, but not from PAH patients. While in rats with experimental PH nintedanib has no effects on the pulmonary vascular pathology, it has favourable effects on RV remodelling.

WNT7A/B promote choroidal neovascularization

Perturbations in WNT signaling are associated with congenital eye disorders, including familial exudative vitreoretinopathy and Norrie disease. More recently, activation of the WNT pathway has also been shown to be associated with age-related macular degeneration (AMD). In this study, we identified that in choroidal neovascular membranes from AMD patients, β-catenin is activated specifically in the vascular endothelium, suggesting that WNT promotes pathologic angiogenesis by directly affecting vascular endothelial cells. WNT7B has been shown to be important during eye development for regression of the fetal hyaloid vasculature. However, it has not yet been established whether WNT7A and/or WNT7B are involved in neovascular AMD pathogenesis. Here, we show that WNT7A and WNT7B increase the proliferation of human dermal microvascular endothelial cells in a dose-dependent manner. Both WNT7A and WNT7B also stimulated vascular sprouting from mouse choroidal explants in vitro. To evaluate in vivo relevance, we generated mice systemically deficient in Wnt7a and/or Wnt7b. Genetic deletion of both Wnt7a and Wnt7b decreased the severity of laser injury-induced choroidal neovascularization (CNV), while individual deletion of either Wnt7a or Wnt7b did not have a significant effect on CNV, suggesting that WNT7A and WNT7B have redundant pro-angiogenic roles in vivo. Cumulatively, these findings identify specific WNT isoforms that may play a pathologic role in CNV as observed in patients with neovascular AMD. Although the source of increased WNT7A and/or WNT7B in CNV requires further investigation, WNT signaling may be a potential target for therapeutic intervention if these results are demonstrated to be relevant in human disease.

Intravitreal implantable magnetic micropump for on-demand VEGFR-targeted drug delivery

In this paper, we propose an intravitreal implantable magnetic micropump integrated with micro check valve capable of on-demand vascular endothelial growth factor receptor (VEGFR)-targeted drug delivery for the treatment of age-related macular degeneration, diabetic retinopathy and other eye pathologies characterized by ocular neoangiogenesis. Precise on-demand drug release is realized by the deflection of the magnetic membrane assembly according to the external magnetic field, and the membrane assembly consists of a thin elastic polydimethylsiloxane (PDMS) membrane and a cylindrical magnetic nanoparticle-PDMS composite block. Additionally, a micro check valve composed of two PDMS layers was integrated into the micropump to realize a diode-like one-directional drug delivery and prevent undesired drug diffusion. For specifically targeting VEGFR and suppression of VEGF-induced proliferation of microvascular endothelial cells, anti-Flt1 gold nanocomplexes are synthesized. In vitro and in vivo experiments and quantitative analysis are carried out in order to verify our proposed concept: precise drug release control according to the external magnetic field, targeting to microvascular endothelial cells, and efficient and on-demand drug delivery from the proposed micropump to the macular area of rabbit's eye.