MiR-150 enhances the motility of EPCs in vitro and promotes EPCs homing and thrombus resolving in vivo

Introduction

Deep venous thrombosis (DVT) is one of the common peripheral vascular diseases. The recruitment and migration of bone marrow-derived endothelial progenitor cells (EPCs) to the sites of venous thrombus are necessary in the process of thrombus organization and recanalization. Our objective was to investigate the functional role of miR-150 in rat EPCs and its potential application in deep venous thrombosis.

Materials and Methods

Rat EPCs were cultured and transfected with miR-150 mimics and inhibitor. Wound healing assay, transwell migration assay and matrigel tube formation assay were performed to elucidate the effect of miR-150 of rat EPCs. Lentiviral construct expressing miR-150 was transfected into EPCs and the EPCs were injected to rat models of DVT. The rats were sacrificed on the day of 7 and 14 after the transplantation and the histological study was performed. Luciferase reporter assay and Western blot were performed to evaluate rat miR-150 regulates the expression of c-Myb.

Results

MiR-150 significantly promoted the migration and tube formation ability of EPCs in vitro and enhanced EPCs’ homing, organization and resolution ability in vivo. Overexpression of miR-150 significantly reduced the protein level of c-Myb and repressed the activity of a luciferase reporter containing both of the two predicted miR-150 binding sites in c-Myb 3’-UTR, indicating that c-Myb may be a miR-150 target gene.

Conclusion

MiR-150 enhanced the migration, tube formation, homing, thrombus recanalization and resolution ability of rat EPCs. Restoring miR-150 in EPCs revealed potential application in DVT therapy.     

Changes and function of circulating endothelial progenitor cells in patients with cerebral aneurysm

Endothelial dysfunction is a trigger for the formation of cerebral aneurysm (CA). The circulating endothelial progenitor cell (EPC) plays an important role in postnatal vasculogenesis and reduction of endothelial injury. In this study, we tested the hypothesis that decreased number and impaired function of circulating EPCs correlate with CA formation in patients. Blood circulating EPCs were identified by flow cytometry. The level of plasma vascular endothelial growth factor (VEGF) was measured by ELISA. Circulating EPCs from patients (n = 27) were cultured in vitro, and the function of EPCs was evaluated by cell migration and senescence-associated β-galactosidase activity. The number of circulating EPCs was significantly decreased in both unruptured and ruptured CA patients compared with healthy control subjects. Impaired migratory capacity and elevated cellular senescence of cultured EPCs were observed in patients with CA (ruptured and unruptured). The percentages of EPC senescence in patients with CAs were significantly and negatively correlated with the number of circulating EPCs. In addition, there were higher levels of plasma VEGF in CA patients compared with healthy control subjects. Our results show that the numbers and functions of circulating EPCs are reduced in patients with CAs. These findings suggest that the decreased number and impaired function of circulating EPCs in CA patients may contribute to the pathophysiological process of aneurysm formation.     

Inhibition of tissue factor by ixolaris reduces primary tumor growth and experimental metastasis in a murine model of melanoma

Melanoma is a highly metastatic cancer and there is strong evidence that the clotting initiator protein, tissue factor (TF), contributes to its aggressive pattern. TF inhibitors may attenuate primary tumor growth and metastasis. In this study, we evaluated the effect of ixolaris, a TF inhibitor, on a murine model of melanoma B16F10 cells. Enzymatic assays performed with B16F10 and human U87-MG tumor cells as the TF source showed that ixolaris inhibits the generation of FX in either murine, human or hybrid FVIIa/TF complexes. The effect of ixolaris on the metastatic potential was further estimated by intravenous injection of B16F10 cells in C57BL/6 mice. Ixolaris (250 μg/kg) dramatically decreased the number of pulmonary tumor nodules (4 ± 1 compared to 47 ± 10 in the control group). Furthermore, a significant decrease in tumor weights was observed in primary tumor growth assays in animals treated with ixolaris (250 μg/kg) from days 3 to 18 after a subcutaneous inoculation of melanoma cells. Remarkably, immunohistochemical analyses showed that inhibition of melanoma growth by ixolaris is accompanied by a significant downregulation of both vascular endothelial growth factor (VEGF) expression and microvascular density in the tumor mass. Our data demonstrate that ixolaris targets B16F10 cell-derived TF, resulting in the reduction of both the primary tumor growth and the metastatic potential of melanoma, as well as the inhibition of tumor angiogenesis. Therefore TF may be a potential target for the treatment of this aggressive malignancy.     

Aging is associated with impaired thrombus resolution in a mouse model of stasis induced thrombosis

Introduction

To evaluate the effects of aging on venous thrombosis.

Material and Methods

Anesthetized male mice (C57BL/6, n = 125) underwent complete inferior vena cava occlusion to produce venous thrombosis. Experimental groups included 11-month-old mice (OLD), 2-month-old mice (YOUNG), and age-matched non-thrombosed controls. Mice were euthanized and the following parameters were evaluated two days post-thrombosis: thrombus mass (grams/cm), vein wall inflammatory cells (cells per 5 high powered fields), active plasma plasminogen activator inhibitor-1 (PAI-1, ng/mL), vein wall P-selectin protein determination by ELISA (pg/mL), circulating plasma microparticles (MPs, MPs/200 µL), MP tissue factor (TF) activity (pM), and in vivo MP re-injection experiments.

Results

Thrombosed OLD mice had greater thrombus mass than YOUNG mice (389 ± 18 vs. 336 ± 14 g × 10^− 4/cm, P < .05). OLD mice had decreased vein wall monocyte, lymphocyte, and total inflammatory cell populations versus YOUNG mice (P < .05). Vein wall P-selectin levels were greater in OLD thrombosed mice versus YOUNG (7306 ± 938 vs. 3805 ± 745 pg/mL, P < .05). Active plasma PAI-1 concentrations were increased in OLD mice versus YOUNG thrombosed animals (20 ± 4 vs. 8 ± 2 ng/mL, P < .05). OLD mice had significantly higher circulating leukocyte-derived MPs versus YOUNG mice (5817 ± 850 vs. 2563 ± 283 MPs/200 μL PPP, P < .01). OLD mice had plasma MPs with increased TF activity versus YOUNG animals post-thrombosis (34 ± 4 vs. 24 ± 2 pM, P < .05). Finally, YOUNG recipient animals, whether re-injected with OLD or YOUNG donor MPs, had a significant increase in thrombus mass versus OLD recipient animals (P < .01).

Conclusion

Aging influenced several circulating and vein wall factors that decreased thrombus resolution in older animals compared to younger ones in our mouse thrombosis model.     

Coronary thrombi neovascularization in patients with ST-elevation myocardial infarction - clinical and angiographic implications

Introduction

Coronary artery thrombosis in ST-elevation myocardial infarction (STEMI) is a dynamic process often preceded by episodes of silent plaque rupture and subocclusive thrombosis. Thrombus organization is achieved by ingrowth of endothelial and smooth muscle cells. Clinical significance and impact of thrombus neovascularization on primary percutaneous coronary intervention (pPCI) outcome remain unclear. Therefore we investigated composition and neovascularization of thrombi aspirated during pPCI and their association with clinical and angiographic parameters of STEMI patients.

Methods

Aspirated thrombi retrieved from 84 STEMI patients were classified as fresh (< 1 day), lytic (1-5 days) or organized (> 5 days). Thrombus neovascularization was evaluated immunohistochemically using CD34, CD31 and VEGF antibodies. CD34 and CD31 immunopositive (CD34/CD31 +) cells were organized as single, clusters and microvessels. VEGF positivity was graded as low or high, based on thrombus surface immunopositive area.

Results

CD34/CD31 + cells were present in 67% of all aspirated thrombi. Thrombus CD34/CD31 positivity was associated with previous history of angina pectoris (χ² = 6.142, p = 0.013) and lower myocardial blush grade (MBG < 3, χ² = 12.602, p < 0.001). Organization of CD34/CD31 + cells showed inverse association with the extent of VEGF positivity (χ² = 10.607, p = 0.005). Fresh thrombi were associated with shorter ischemic time (U = 237.5, p = 0.002) and MBG 3 (χ² = 6.379, p = 0.012).

Conclusions

Older thrombus age and neovascularization are associated with suboptimal myocardial perfusion in STEMI patients. Thrombus VEGF expression is inversely associated with degree of CD34 + cell organization. Therefore, neovascularization of aspirated thrombi may indicate the duration of thrombosis, coronary microcirculation status and outcome in STEMI patients.     

Prognostic significance of adhesion molecules (sICAM-1, sVCAM-1) and VEGF in colorectal cancer patients

Introduction

Adhesion molecules take part in the interaction between host cells and cancer cells. In the current study the relationship between the soluble adhesion molecules sICAM-1 and sVCAM-1 and proangiogenic factor VEGF in colorectal cancer progression were measured.

Materials and methods

The study group consisted of 46 patients with colorectal carcinoma (classified due to TNM classification) and 40 controls. sICAM-1, sVCAM-1 and VEGF plasma concentration were measured by enzyme-linked immunosorbent assay (ELISA).

Results

All measured parameters levels were increased significantly in patients with colorectal cancer in comparison to controls (p < 0.001). sICAM-1, sVCAM-1 and VEGF increased significantly due to colorectal cancer progression. There was a positive correlation between sICAM-1 and sVCAM-1 in all study groups.

Conclusions

Our results demonstrated in CRC patients significantly increased levels of soluble adhesion molecules (VCAM-1 and sICAM-1) and angiogenic factor (VEGF) as compared to control group. The dynamics of these molecules showed the growing tendency along with tumor size and metastasis formation.     

Effect of resection of localized pancreaticobiliary adenocarcinoma on angiogenic markers and tissue factor related pro-thrombotic and pro-angiogenic activity

In this study, 52 patients were studied to elucidate the relative impact of resection of localized pancreaticobiliary adenocarcinoma (PBC) on circulating factors of tumour-associated angiogenesis e.g. tissue factor bearing microparticles (TFMP) and vascular endothelial growth factor (VEGF) and their clinicopathological significance to angiogenesis markers in cancer tissue from PBC patients. Angiogenesis array analysis on serum samples revealed that surgical resection of tumour lesion in PBC patients affects the levels of a panel of angiogenesis-related molecules, including VEGF that was verified by ELISA to significantly reduce (median & IQR: 1003(369-2000) vs. 457(159-834) pg/ml; p < 0.05). Correspondingly, a significant decrease in the angiogenic activity (decreased capillary tube formation; p < 0.05) of serum samples after the surgery was also found. Despite a decrease in number of circulating TFMP after surgery, this did not reach statistical significance; there was a significant reduction in pro-coagulant activity (prolonged prothrombin time, p < 0.001) post-operatively. In addition, the activity of total microparticles (MP activity assay, p < 0.05) was decreased significantly. Immunohistochemical staining of tumour tissue revealed a strong correlation between the microvessel density (MVD) and VEGF expression. Also, higher levels of circulating TFMP or TF related activity (prothrombin time) correlated significantly with TF expression and MVD on tumour tissues from PBC patients. These findings suggest that in pancreaticobiliary adenocarcinoma TF related angiogenesis drivers are equally significant to VEGF ones, raising the clinical question of whether the effectiveness of angiogenesis targeting studies could be improved through the ‘dual’ targeting of these pathways in PBC.     

Treatment with everolimus is associated with a procoagulant state

Introduction

Renal transplant recipients are at increased risk of venous thromboembolic events, which is in part caused by their treatment with maintenance immunosuppressive drugs. Because we observed an increased incidence of venous thromboembolic events in renal transplant recipients treated with the mTOR inhibitor (mTORi) everolimus, we aimed to identify prothrombotic mechanisms of this immunosuppressive drug.

Materials and Methods

In a single center study, nested in a multi-center randomized controlled trial, we measured parameters of coagulation, anti-coagulation and fibrinolysis in renal transplant recipients, receiving the mTORi everolimus (n = 16, mTOR group) and compared them to a similar patient group, receiving a calcineurin inhibitor and/or mycophenolate sodium (n = 20, non-mTOR group). All patients were at least 6 months following transplantation with a stable transplant function.

Results

The use of an mTORi was associated with significantly higher levels of von Willebrand factor, prothrombin fragment 1 + 2, thrombin-activatable fibrinolysis inhibitor and plasminogen activator inhibitor-1 as compared to a non-mTORi based immunosuppressive regimen.

Conclusions

Treatment with an mTORi leads to increased endothelial activation, thrombin formation and impaired fibrinolysis in renal transplant recipients. This suggests an increased risk of thrombotic events in renal transplant recipients treated with mTOR inhibitors. A prospective study to establish the precise risk of thrombotic events in these patients is urgently needed.     

Male mice have increased thrombotic potential: sex differences in a mouse model of venous thrombosis

Introduction

Our objectives were to characterize sex differences during venous thrombosis, using the electrolytic inferior vena cava model of the disease.

Materials and methods

Male and female C57BL/6 mice (6-8 weeks) underwent inferior vena cava thrombosis. Time points included 6 hours, day 2, day 6, and day 14 post surgery, along with surgically naïve true controls and surgical shams. Analyses included thrombus weight, vein wall morphometrics, vein wall protein and gene expression for P-selectin, interleukin-1β, and tumor necrosis factor-α; hematology, soluble P-selectin, and plasma microparticle tissue factor activity assays.

Results

Male venous thrombi were significantly larger than females at days 2 (13.1 ± 1.0 vs. 6.8 ± 0.5 × 10^-3 grams, p < 0.01), 6 (10.4 ± 0.8 vs. 5.4 ± 0.5 × 10^-3 grams, p < 0.01) and 14 (6.3 ± 0.5 vs. 4.1 ± 0.3 × 10^-3 grams, p < 0.01). Both male and female mice exhibited significantly increased vein wall P-selectin at 6 hours, vs. true controls (p < 0.05). Males had increased vein wall interleukin-1β, versus females, at 6 hours (180.926 ± 24.596 vs. 60.417 ± 10.478 pg/mL, p < 0.05) and day 6 (76.966 ± 13.081 vs. 33.834 ± 4.198 pg/mL, p < 0.01). Males showed decreased tumor necrosis factor-α expression (-66 %) at 6 hours. Females had increased tumor necrosis factor-α expression at 6 hours (+ 541%) and day 6 (+ 539%). Both sexes demonstrated decreased peripheral platelets at 6 hours (p < 0.05), coinciding with thrombogenesis. Plasma P-selectin increased in both sexes, versus controls, through day 6 (p < 0.05).

Conclusions

Males had significantly larger venous thrombi than females. Sex differences in vascular anatomy and response to inflammation may influence thrombus formation in our mouse thrombosis model.     

Anti-thrombosis effect of LRRFIP1 shRNA lentivirus in a mouse model of deep vein thrombosis

Introduction

Deep vein thrombosis (DVT) is one of the common complications of orthopedic surgery. Low molecular weight heparin (LMWH) is a usually used agent for DVT, but it would increase the risk of bleeding. LRRFIP1 has been shown to play an important role in the formation of thrombosis. Therefore, we investigated the effect of LRRFIP1 shRNA lentivirus on DVT in mice.

Materials and Methods

Lentiviral Vectors carrying LRRFIP1 shRNA were constructed and transfected into cultured mouse bone marrow cells (BMCs). Male ICR mice were irradiated with a single dose of 9.5 Gy and then were injected with different agents through the tail vein. Stasis venous thrombosis was induced by inferior vena cava (IVC) ligation. Mice were sacrificed on the 1st, 3rd and 7th day post operation and the thrombi were removed, blotted the excess blood on it with filter paper and immediately weighed. P-selectin and d-Dimer were determined by enzyme-linked immunosorbent assay (ELISA).

Results

LRRFIP1 shRNA significantly suppressed the expression of LRRFIP1 in the thrombi. In contrast, low molecular weight heparin (LMWH) and negative shRNA exhibited little effect on the expression of LRRFIP1. LRRFIP1 shRNA, LMWH and negative shRNA inhibited the thrombus formation in vivo significantly. The plasma P-selectin and d-Dimer levels were significantly increased after IVC ligation. LRRFIP1 shRNA significantly decreased the plasma P-selectin and d-Dimer levels. However, LMWH and negative shRNA showed little effects on the levels of plasma P-selectin and d-Dimer.

Conclusion

LRRFIP1 shRNA might represent a promising prevention strategy for DVT.     

Role of VEGF and matrix metalloproteinase‐9 in peritumoral brain edema associated with supratentorial benign meningiomas

Accumulating evidence indicates that VEGF and matrix metalloproteinase‐9 (MMP‐9) play a central role in the development of peritumoral brain edema (PTBE) associated with human brain tumors. However, the roles of these proteins, particularly of MMP‐9, in PTBE associated with benign meningiomas have not been elucidated. We investigated the association between clinical features and biological factors, such as VEGF and MMP‐9, and the incidence of PTBE and edema index (EI) in 60 patients with benign meningiomas. In this study, supratentorial lesions were examined for evaluating the extent of PTBE in the surrounding normal brain tissue. VEGF and MMP‐9 expression was immunohistochemically examined. Multivariate analysis revealed that the presence of pial blood supply (odds ratio [OR] 12.250; P = 0.0096) and VEGF (OR 7.683; P = 0.0155), but not MMP‐9 (OR 1.178; P = 0.8113), expression are significant factors that independently predict the incidence of PTBE and influence EI. VEGF (P = 0.0397) and MMP‐9 (P = 0.0057) expression correlates with the presence of pial blood supply. Moreover, tumors with high VEGF and MMP‐9 expression had higher EIs than those with high expression of either (P = 0.030). Our findings suggest that MMP‐9 expression was positively related to VEGF expression and pial blood supply and promoted the occurrence of PTBE by inducing the disruption of the arachnoid membrane and formation of pial blood supply.     

脑动静脉畸形破裂出血病人中Ang1、Ang2、VEGF、LP的表达和意义

目的研究血管生成素1(angiopoietin 1,Ang1)、血管生成素2(angiopoietin 2,Ang2)、血管内皮生长因子(vascular-endothelial growth factor,VEGF)及瘦素(leptin,LP)在脑动静脉畸形(AVM)破裂出血病人中的表达和意义。方法收集手术切除的AVM标本28例,将14例有近期破裂出血史的病人作为AVM出血组,14例无明显破裂出血史的病人作为AVM未出血组,采用免疫组织化学法检测2组标本Ang1、Ang2、VEGF及LP的表达,并进行统计分析。结果免疫组织化学法显示:Ang1、Ang2、VEGF及LP在2组血管内皮细胞中均有阳性表达;与AVM未出血组比较,AVM出血组的血管内皮细胞中Ang2、VEGF、LP表达显著增强(均P<0.01),而Ang1的表达较弱(P>0.05)。结论 Angl、Ang2、VEGF及LP在AVM的畸形血管中存在特异性表达,尤其是Ang2、VEGF及LP可能与AVM的进展及破裂出血有密切关系。     

Elevated platelet angiostatin and circulating endothelial microfragments in idiopathic pulmonary arterial hypertension: A preliminary study

Introduction

Idiopathic pulmonary arterial hypertension (IPAH) is characterized by pulmonary arteriolar narrowing and degeneration associated with in situ thrombosis. We hypothesized that microvascular endothelial injury and apoptosis may be an initiating mechanism in IPAH. Endothelial apoptosis generates endothelial microfragments (EMF), which can activate platelets. Platelets release both VEGF and angiostatin, which depending the balance can inhibit or induce endothelial apoptosis, respectively.

Materials and Methods

We measured EMFs from blood of IPAH patients as index of endothelial cell apoptosis/injury and levels of pro- and anti- EC apoptotic factors found in platelets. EMFs and platelets in blood samples from control subjects and patients with IPAH were measured using a 4-color flow cytometry protocol, and platelet levels of VEGF and angiostatin were determined by ELISAs and immunoblotting.

Results

Compared to controls, IPAH patients exhibited higher numbers of circulating EMFs and more activated/apoptotic platelets. IPAH patients also exhibited higher levels of platelet angiostatin; however, no significant difference was detected in platelet VEGF levels between the two groups.

Conclusions

These results are consistent with an increase in EC dysfunction in patients with IPAH, possibly contributing to the progression of IPAH and its associated thrombosis.     

Altered serum levels of vascular endothelial growth factor and glial-derived neurotrophic factor but not fibroblast growth factor-2 in treatment-naive children with attention deficit/hyperactivity disorder

Abstract

Background and aim: Recent evidence suggests that growth factors might be involved in the pathophysiology of attention deficit hyperactivity disorder (ADHD). The aim of this study was to determine whether serum levels of brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF), neurotrophin-3 (NT-3), nerve growth factor (NGF), fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF) were altered in children with ADHD.

Methods: Serum levels of BDNF, GDNF, NT-3, NGF, VEGF and FGF-2 were analyzed in 49 treatment- naive children with ADHD and age, gender matched 36 healthy controls using enzyme-linked immunosorbent assay. ADHD symptoms were scored by Du Paul ADHD Rating Scale and Strengths and Difficulties Questionnaire.

Results: We found that serum VEGF levels were significantly lower (p?<?0.001) and GDNF levels were significantly higher in ADHD group compared to control group (p?=?0.003). However, we found no correlations between ADHD symptoms and serum VEGF or GDNF levels. Furthermore, we observed no significant alterations in serum BDNF, NT-3, NGF, FGF-2 levels in children with ADHD.

Conclusion: To our knowledge, the present study is the first to examine serum VEGF and FGF-2 levels in children with ADHD. Our results indicate that VEGF and GDNF might be involved in the etiology of ADHD. Further studies are required to determine the role of growth factors in the etiology and consequently in the treatment of ADHD.