Decreased Microvessel Density Research Articles

Monitoring imatinib decreasing pericyte coverage and HIF-1α level in a colorectal cancer model by an ultrahigh-field multiparametric MRI approach

BackgroundExcessive pericyte coverage promotes tumor growth, and a downregulation may solve this dilemma. Due to the double-edged sword role of vascular pericytes in tumor microenvironment (TME), indiscriminately decreasing pericyte coverage by imatinib causes poor treatment outcomes. Here, we optimized the use of imatinib in a colorectal cancer (CRC) model in high pericyte-coverage status, and revealed the value of multiparametric magnetic resonance imaging (mpMRI) at 9.4T in monitoring treatment-related changes in pericyte coverage and the TME.MethodsCRC xenograft models were evaluated by histological vascular characterizations and mpMRI. Mice with the highest pericyte coverage were treated with imatinib or saline; then, vascular characterizations, tumor apoptosis and HIF-1α level were analyzed histologically, and alterations in the expression of Bcl-2/bax pathway were assessed through qPCR. The effects of imatinib were monitored by dynamic contrast-enhanced (DCE)-, diffusion-weighted imaging (DWI)- and amide proton transfer chemical exchange saturation transfer (APT CEST)-MRI at 9.4T.ResultsThe DCE- parameters provided a good histologic match the tumor vascular characterizations. In the high pericyte coverage status, imatinib exhibited significant tumor growth inhibition, necrosis increase and pericyte coverage downregulation, and these changes were accompanied by increased vessel permeability, decreased microvessel density (MVD), increased tumor apoptosis and altered gene expression of apoptosis-related Bcl-2/bax pathway. Strategically, a 4-day imatinib effectively decreased pericyte coverage and HIF-1α level, and continuous treatment led to a less marked decrease in pericyte coverage and re-elevated HIF-1α level. Correlation analysis confirmed the feasibility of using mpMRI parameters to monitor imatinib treatment, with DCE-derived Ve and Ktrans being most correlated with pericyte coverage, Ve with vessel permeability, AUC with microvessel density (MVD), DWI-derived ADC with tumor apoptosis, and APT CEST-derived MTRasym at 1 µT with HIF-1α.ConclusionsThese results provided an optimized imatinib regimen to achieve decreasing pericyte coverage and HIF-1α level in the high pericyte-coverage CRC model, and offered an ultrahigh-field multiparametric MRI approach for monitoring pericyte coverage and dynamics response of the TME to treatment.

DIPG-68. FLAG-003, A NOVEL, DUAL-ACTION THERAPEUTIC, DEMONSTRATES IN-VITRO ACTIVITY AGAINST DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG)

Abstract BACKGROUND FLAG-003 is a novel, rationally-designed, CNS-penetrant, dual-acting agent that inhibits receptor tyrosine kinase activity and microtubule polymerization. Preclinical studies in glioblastoma (GBM) demonstrated decreased cell viability, reduced tumor volumes, decreased microvessel density, and increased survival. Remarkably, using an orthotopic DIPG model, all mice treated with FLAG-003 at 275 mg/day/orally displayed tumor regression, tumor reduction and increased survival. Given the significant preclinical activity against DIPG, as well as the known heterogeneity of DIPG, we evaluated the efficacy of FLAG-003 in vitro against a panel of DIPG cell lines. METHODS Eight DIPG cell lines hosted at the Center for Patient Derived Models (CPDM) were selected based on genomic and growth characteristics. Cells were plated on 384-well plates; 24 hours after plating, cells were dosed with 1nM to 50uM (12 step log curve) FLAG-003. Three technical replicates were performed per step. Controls consisted of positive kill control Staurosporine, negative growth control of 0.5% DMSO, or no DMSO for baseline growth. ONC201, a small molecule antagonist of DRD2/3 currently in clinical trials for DIPG, was used as a comparator. We also evaluated two GBM patient-derived models. RESULTS FLAG-003 demonstrated efficacy in all DIPG and GBM cell lines tested with IC50 less than 100nM. FLAG-003 was more potent compared to ONC201 in all models evaluated. CONCLUSIONS Single agent FLAG-003 demonstrated activity against a panel of DIPG cell lines. As next steps, we are exploring the efficacy of this drug in orthotopic DIPG PDX models. Continued development of FLAG-003 as a potential therapeutic candidate for DIPG patients is warranted and underway.

Wrecking neutrophil extracellular traps and antagonizing cancer-associated neurotransmitters by interpenetrating network hydrogels prevent postsurgical cancer relapse and metastases

Tumor-promoting niche after incomplete surgery resection (SR) can lead to more aggressive local progression and distant metastasis with augmented angiogenesis-immunosuppressive tumor microenvironment (TME). Herein, elevated neutrophil extracellular traps (NETs) and cancer-associated neurotransmitters (CANTs, e.g., catecholamines) are firstly identified as two of the dominant inducements. Further, an injectable fibrin-alginate hydrogel with high tissue adhesion has been constructed to specifically co-deliver NETs inhibitor (DNase I)-encapsulated PLGA nanoparticles and an unselective β-adrenergic receptor blocker (propranolol). The two components (i.e., fibrin and alginate) can respond to two triggers (thrombin and Ca2+, respectively) in postoperative bleeding to gelate, shaping into an interpenetrating network (IPN) featuring high strength. The continuous release of DNase I and PR can wreck NETs and antagonize catecholamines to decrease microvessel density, blockade myeloid-derived suppressor cells, secrete various proinflammatory cytokines, potentiate natural killer cell function and hamper cytotoxic T cell exhaustion. The reprogrammed TME significantly suppress locally residual and distant tumors, induce strong immune memory effects and thus inhibit lung metastasis. Thus, targetedly degrading NETs and blocking CANTs enabled by this in-situ IPN-based hydrogel drug depot provides a simple and efficient approach against SR-induced cancer recurrence and metastasis.

AZGP1 deficiency promotes angiogenesis in prostate cancer

BackgroundLoss of AZGP1 expression is a biomarker associated with progression to castration resistance, development of metastasis, and poor disease-specific survival in prostate cancer. However, high expression of AZGP1 cells in prostate cancer has been reported to increase proliferation and invasion. The exact role of AZGP1 in prostate cancer progression remains elusive.MethodAZGP1 knockout and overexpressing prostate cancer cells were generated using a lentiviral system. The effects of AZGP1 under- or over-expression in prostate cancer cells were evaluated by in vitro cell proliferation, migration, and invasion assays. Heterozygous AZGP1± mice were obtained from European Mouse Mutant Archive (EMMA), and prostate tissues from homozygous knockout male mice were collected at 2, 6 and 10 months for histological analysis. In vivo xenografts generated from AZGP1 under- or over-expressing prostate cancer cells were used to determine the role of AZGP1 in prostate cancer tumor growth, and subsequent proteomics analysis was conducted to elucidate the mechanisms of AZGP1 action in prostate cancer progression. AZGP1 expression and microvessel density were measured in human prostate cancer samples on a tissue microarray of 215 independent patient samples.ResultNeither the knockout nor overexpression of AZGP1 exhibited significant effects on prostate cancer cell proliferation, clonal growth, migration, or invasion in vitro. The prostates of AZGP1−/− mice initially appeared to have grossly normal morphology; however, we observed fibrosis in the periglandular stroma and higher blood vessel density in the mouse prostate by 6 months. In PC3 and DU145 mouse xenografts, over-expression of AZGP1 did not affect tumor growth. Instead, these tumors displayed decreased microvessel density compared to xenografts derived from PC3 and DU145 control cells, suggesting that AZGP1 functions to inhibit angiogenesis in prostate cancer. Proteomics profiling further indicated that, compared to control xenografts, AZGP1 overexpressing PC3 xenografts are enriched with angiogenesis pathway proteins, including YWHAZ, EPHA2, SERPINE1, and PDCD6, MMP9, GPX1, HSPB1, COL18A1, RNH1, and ANXA1. In vitro functional studies show that AZGP1 inhibits human umbilical vein endothelial cell proliferation, migration, tubular formation and branching. Additionally, tumor microarray analysis shows that AZGP1 expression is negatively correlated with blood vessel density in human prostate cancer tissues.ConclusionAZGP1 is a negative regulator of angiogenesis, such that loss of AZGP1 promotes angiogenesis in prostate cancer. AZGP1 likely exerts heterotypical effects on cells in the tumor microenvironment, such as stromal and endothelial cells. This study sheds light on the anti-angiogenic characteristics of AZGP1 in the prostate and provides a rationale to target AZGP1 to inhibit prostate cancer progression.

Effects of Glutamine Synthetase on Neovascularization in Glioma: In Vivo MR Vessel Size Imaging and Histology.

Glutamine Synthetase (GS) could induce vascular sprouting through the improvement of endothelial cell migration in inflammatory diseases. MR vessel-size imaging has been proposed as a valuable approach for visualizing the underlying angiogenic processes in the brain. This study aims to investigate the role of GS in the neovascularization of gliomas through the utilization of MR vessel-size imaging and histopathological techniques. In this exploratory animal study, we randomly divided the C6 glioma rat models into a control group and an L-methionine sulfoximine (MSO) treatment group. Daily intraperitoneal injections were administered for three consecutive days, starting from day 10 following the implantation of C6 glioma cells in rats. Subsequently, MR vessel size imaging was conducted using a BRUKER 7 T/200 mm MRI scanner, and the MRI results were validated through histopathological examination. A significant decrease in microvessel density was observed in both the tumor periphery and center areas in the MSO treatment group compared to that in the control group. The mean vessel diameter (mVD) and vessel size index (VSI) did not exhibit significant changes compared to the control group. Moreover, the staining intensity of platelet endothelial cell adhesion molecule-1 (CD31) and GS in the tumor periphery was significantly decreased in the MSO treatment group. Additionally, the MSO treatment demonstrated a substantial inhibition of tumor growth. GS inhibitors significantly reduced angiogenesis in the periphery area of C6 glioma, exerting an inhibitory effect on tumor progression. Thus, GS inhibitors could be potential therapeutic agents for treating glioma. Additionally, in vivo MR vessel size imaging detects changes in vascularrelated parameters after tumor treatment, making it a promising method for detecting neovascularization in glioma.</p>.

Neutrophil extracellular traps promote angiogenesis in gastric cancer

Although antiangiogenic therapy has been used in gastric cancer, disease progression due to drug resistance remains common. Neutrophils play an important role in the occurrence and progression of cancer via neutrophil extracellular traps (NETs). However, few studies have investigated angiogenic regulation in gastric cancer. We aimed to determine the role of NETs in promoting angiogenesis in gastric cancer. Multiple immunohistochemical staining was used to analyze the spatial distribution of NETs and microvessels in patient tissue samples. A mouse subcutaneous tumor model was established to determine the effect of NETs on tumor growth, and changes in microvessel density were observed via immunohistochemical staining. We screened differentially expressed proteins in HUVECs stimulated by NETs via proteomics. Cell Counting Kit-8, EdU labeling, and tubule formation assays were used to verify the effect of NETs on HUVEC proliferation, migration, and tubule formation. Blocking NETs, which was related to decreased microvessel density, significantly inhibited tumor growth in the murine subcutaneous tumor model. Compared with those of the control group, tumor volume and mass among mice in the inhibition group decreased by 61.3% and 77.9%, respectively. The NET-DNA receptor CCDC25 was expressed in HUVECs, providing a platform for NETs to promote HUVEC proliferation, migration, and tubulation. In an in vitro rat aortic explant model, NETs induced HUVEC proliferation, survival, and chemotaxis, which were not significantly different from those observed in the VEGF stimulation group. Our results confirm that NETs promote angiogenesis in gastric cancer, providing a theoretical basis for identifying new anti-vascular therapeutic targets.Graphical 1Sf8jYXSegxbAWaiHcqZYYVideo

Xanthine oxidase mediates stress-induced Alzheimer's Disease progression in triple transgenic mice

Alzheimer’s disease (AD) is a progressive disorder associated with oxidative stress and neuroinflammation promoting the deposition of β-amyloid (Aβ) plaques and neurofibrillary tau tangles. Xanthine oxidoreductase (XOR) is a molybdoenzyme responsible for the metabolism of hypoxanthine and xanthine to uric acid. Under inflammatory conditions, high plasma uric acid levels upregulate XOR leading to increased free radical production and promoting cerebrovascular dysfunction. Chronic stress induces cerebrovascular dysfunction and cognitive decline in C57BL/6 mice mediated by XOR, suggesting a link between chronic stress and dementia. However, there is no published data that link XOR and stress to AD pathology and progression. Thus, we tested the hypothesis that chronic stress accelerates AD pathology via the XOR pathway.At 4 months of age, male and female WT (B6129) and triple transgenic (3xTg) AD mice underwent the Unpredictable Chronic Mild Stress (UCMS) model paradigm for 8 weeks. Mice were euthanized at two different time points at 6 and 9 months of age for both control and UCMS groups, and the tissues were harvested. Brain tissue lysates were homogenized, and the total tau and XOR protein was quantified using Western blot and normalized to β-actin. Aβ (1-40) levels in mouse brain homogenates using Aβ ELISA kit. Immunohistochemical staining was performed to study the colocalization of XO and CD-31 in WT and 3xTg coronal sections and analyzed using Fiji ImageJ.UCMS accelerated Aβ(1-40) expression levels (p&lt;0.05) at both 6 and 9 months of age in the 3xTg mice. Tau levels were significantly increased at 6 months of age (p&lt;0.05), but showed no significant differences in protein expression at 9 months of age. XOR levels were significantly upregulated (p&lt;0.05) in the UCMS group at 6 months of age. At 9 months of age, no significant differences were seen in XOR expression across groups indicating that mice taken off from the UCMS protocol had normal levels of XOR due to reduced oxidative stress buildup in the brain. At 6 months of age, a significant decrease in microvessel density (p&lt;0.05) was seen in 3xTg-UCMS mice vs 3xTg controls, with no significant differences seen at 9 months of age, indicating the role of chronic stress on the decline in cerebrovascular functions. Thus, the study establishes the premise that chronic stress mediated by XOR accelerates AD pathology in our early-onset AD model. NIH, NINDS BINP R01 NS117754, Psychosocial Stress-Induced Vascular Contributions to Cognitive Impairment and Alzheimer's Disease: The Role of Xanthine Oxidase This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Everolimus downregulates STAT3/HIF-1α/VEGF pathway to inhibit angiogenesis and lymphangiogenesis in TP53 mutant head and neck squamous cell carcinoma (HNSCC).

TP53 mutant head and neck squamous cell carcinoma (HNSCC) patients exhibit poor clinical outcomes with 50-60% recurrence rates in advanced stage patients. In a recent phase II clinical trial, adjuvant therapy with everolimus (mTOR inhibitor) significantly increased 2-year progression-free survival in p53 mutated patients. TP53-driven mTOR activation in solid malignancies causes upregulation of HIF-1α and its target, downstream effector VEGF, by activating STAT3 cell signaling pathway. Here, we investigated the effects of everolimus on the STAT3/HIF-1α/VEGF pathway in TP53 mutant cell lines and xenograft models. Treatment with everolimus significantly inhibited cell growth in vitro and effectively reduced the growth of TP53 mutant xenografts in a minimal residual disease (MRD) model in nude mice. Everolimus treatment was associated with significant downregulation of STAT3/HIF-1α/VEGF pathway in both models. Further, treatment with everolimus was associated with attenuation in tumor angiogenesis and lymphangiogenesis as indicated by decreased microvessel density of vascular and lymphatic vessels in HN31 and FaDu xenografts. Everolimus downregulated the STAT3/HIF-1α/VEGF pathway to inhibit growth and in vitro tube formation of HMEC-1 (endothelial) and HMEC-1A (lymphatic endothelial) cell lines. Our studies demonstrated that everolimus inhibits the growth of TP53 mutant tumors by inhibiting angiogenesis and lymphangiogenesis through the downregulation of STAT3/HIF-1α/VEGF signaling.

Increased vulnerability to atrial and ventricular arrhythmias caused by different types of inhaled tobacco or marijuana products

The emergence of a plethora of new tobacco products marketed as being less harmful than smoking, such as electronic cigarettes and heated tobacco products, and the increased popularity of recreational marijuana have raised concerns about the potential cardiovascular risk associated with their use. The purpose of this study was to investigate whether the use of novel tobacco products or marijuana can cause the development of proarrhythmic substrate and eventually lead to arrhythmias. Rats were exposed to smoke from tobacco, marijuana, or cannabinoid-depleted marijuana, to aerosol from electronic cigarettes or heated tobacco products, or to clean air once per day for 8 weeks, following by assays for blood pressure, cardiac function, ex vivo electrophysiology, and histochemistry. The rats exposed to tobacco or marijuana products exhibited progressively increased systolic blood pressure, decreased cardiac systolic function with chamber dilation, and reduced overall heart rate variability, relative to the clean air negative control group. Atrial fibrillation and ventricular tachycardia testing by ex vivo optical mapping revealed a significantly higher susceptibility to each, with a shortened effective refractory period and prolonged calcium transient duration. Histological analysis indicated that in all exposure conditions except for air, exposure to smoke or aerosol from tobacco or marijuana products caused severe fibrosis with decreased microvessel density and higher level of sympathetic nerve innervation. These pathophysiological results indicate that tobacco and marijuana products can induce arrhythmogenic substrates involved in cardiac electrical, structural, and neural remodeling, facilitating the development of arrhythmias.

Vascular regression in the kidney: changes in 3D vessel structure with time post-irradiation.

Though angiogenesis has been investigated in depth, vascular regression and rarefaction remain poorly understood. Regression of renal vasculature accompanies many pathological states such as diabetes, hypertension, atherosclerosis, and radiotherapy. Radiation decreases microvessel density in multiple organs, though the mechanism is not known. By using a whole animal (rat) model with a single dose of partial body irradiation to the kidney, changes in the volume of renal vasculature were recorded at two time points, 60 and 90 days after exposure. Next, a novel vascular and metabolic imaging (VMI) technique was used to computationally assess 3D vessel diameter, volume, branch depth, and density over multiple levels of branching down to 70 µm. Four groups of rats were studied, of which two groups received a single dose of 12.5 Gy X-rays. The kidneys were harvested after 60 or 90 days from one irradiated and one non-irradiated group at each time point. Measurements of the 3D vasculature showed that by day-90 post-radiation, when renal function is known to deteriorate, total vessel volume, vessel density, maximum branch depth, and the number of terminal points in the kidneys decreased by 55%, 57%, 28%, and 53%, respectively. Decreases in the same parameters were not statistically significant at 60 days post-irradiation. Smaller vessels with internal diameters of 70-450 µm as well as large vessels of diameter 451-850 µm, both decreased by 90 days post-radiation. Vascular regression in the lungs of the same strain of irradiated rats has been reported to occur before 60 days supporting the hypothesis that this process is regulated in an organ-specific manner and occurs by a concurrent decrease in luminal diameters of small as well as large blood vessels.

Treatment of malignant pleural effusion in non-small cell lung cancer with VEGF-directed therapy

Background Vascular endothelial growth factor (VEGF) is a critical regulator of malignant pleural effusion (MPE) in non-small-cell lung cancer (NSCLC). Bevacizumab (BEV) and apatinib (APA) are novel VEGF blockers that inhibit lung cancer cell proliferation and the development of pleural effusion. Methods In this study, we established Lewis lung cancer (LLC) xenograft mouse models to compare the therapeutic effect of APA and BEV in combination with cisplatin (CDDP) against MPE. The anti-tumour and anti-angiogenic effects of this combination therapy were evaluated by 18F-FDG PET/CT imaging, TUNEL assay and Immunohistochemistry. Results The triple drug combination significantly prolonged the overall survival of the tumour-bearing mice by reducing MPE and glucose metabolism and was more effective in lowering VEGF/soluble VEGFR-2 levels in the serum and pleural exudates compared to either of the monotherapies. Furthermore, CDDP + APA + BEV promoted in vivo apoptosis and decreased microvessel density. Conclusions Mechanistically, LLC-induced MPE was inhibited by targeting the VEGF-MEK/ERK pathways. Further studies are needed to establish the synergistic therapeutic effect of these drugs in NSCLC patients with MPE. KEY MESSAGES Combined treatment of MPE with apatinib, bevacizumab and cisplatin can prolong the survival time of mice, reduce the content of MPE, decrease the SUVmax of thoracic tumour tissue, down-regulate the content of VEGF and sVEGFR-2 in serum and pleural fluid, and promote the apoptosis of tumour cells. Angiogenesis and MPE formation can be inhibited by down-regulation of HIF-1α, VEGF, VEGFR-2, MEK1 and MMP-2 molecular signalling pathway proteins.

Inhibitory effects of icotinib combined with antiangiogenic drugs in human non-small cell lung cancer xenograft models are better than single target drugs.

BackgroundThis study aimed to evaluate the inhibitory effects and potential mechanisms of icotinib combined with antiangiogenic drugs on lung adenocarcinoma in vivo.MethodsA total of 72 mouse xenograft models established with human lung adenocarcinoma cells (HCC827) were randomly divided into six groups, including control, icotinib (Ic), bevacizumab (Bev), recombinant human endostatin (En), Ic + Bev and Ic + En groups. Mouse weights and tumor volumes were measured regularly. Half of the nude mice in each group were sacrificed after 16 days of drug treatment. The remaining animals were observed for another 16 days without drug supply. Immunohistochemical staining was performed to detect microvessel density in tumor heart, liver, brain specimens from the nude mice and Ki67 expression. Differential expression of vascular endothelial growth factor (VEGFA) in tumor tissue specimens was determined by ELISA and Western blot.ResultsThe results showed that the combined drugs inhibited tumor growth more substantially compared with single drugs, without increasing the toxic effects. The antiangiogenesis effect of the combination was better than that of single drug treatment. In addition, both types of targeted drugs and combination medication not only significantly reduced microvessel density in the tumor tissue itself, but also had a certain impact on decreasing microvessel density in the liver. The combination decreased VEGFA and Ki‐67 amounts significantly more than icotinib or endostatin as a monotherapy.ConclusionsIcotinib combined with bevacizumab or rh‐endostatin has a stronger inhibitory effect on tumor growth than single‐target drug in vivo, with no additional side effects.

Study on Damage of Myocardial Tissue Under Low-Dose Heart Irradiation in Rats

<h3>Purpose/Objective(s)</h3> Radiation induced heart damage (RIHD) is an important late toxicity of thoracic radiotherapy, which is closely relating with the fractional dose of radiotherapy. With the wide application of stereotactic body radiotherapy and hyperfractionated radiotherapy, the single dose and biological equivalent dose (BED) of the heart of exposure have increased when comparing with conventionally fractionated radiotherapy. However, the mean cardiac dose (MHD), which is frequently used clinically to ensure the control of cardiac damage, is continuously decreased to 26 Gy, 20 Gy, etc. There is still controversy about how much dose could control heart radiation damage better. In this study, 15 Gy/3f low-dose irradiation of the whole heart of rats, which BED is similar with 24 Gy/12f irradiation, is selected to explore the possibility and characteristics of RIHD occurrence, and explore the effect of combined recombinant human endostatin on myocardial damage. <h3>Materials/Methods</h3> 75 SD adult male rats were randomly divided into control (C group, E group, MHD₂₅ group) and experimental group (MHD₁₅ group, MHD_15+E group). The irradiation method was single dose 5 Gy/f, once per day, continuous irradiation 3d or 5d. After the irradiation, the E group and MHD_15+E group were given recombinant human endostatin 6mg/kg intraperitoneal injection, once per day, for 14 consecutive days. At 1, 3, and 6 months after intervention, 5 rats in each group were randomly selected for HE, Masson, and immunohistochemical CD34 microvessel staining, and Western Blot detected mitochondrial Complex II protein expression. Two-way ANOVA statistics. <h3>Results</h3> Compared with C group, edema and disorder of myocardial cells, infiltration of inflammatory cells and decrease of microvessel density was observed at 1 month after intervention in HE staining in MHD₁₅ and MHD_15+E group. The expression of complex II protein decreased at 6 months, and the degree of decrease was less than that in MHD₂₅ group (positive control group) (<i>P</i> < 0.05). The microvessel density was further decreased compared with 1 month. Myocardial fibrosis induced by 15 Gy/3f low-dose irradiation of SD rat heart mainly occurred at 6 months, which observed myocardial cord or reticular fibrosis and collagen deposition. Semi-quantitative analysis of myocardial collagen volume fraction CVF significantly increased (<i>P</i> < 0.001) in HE and Masson staining. Details is shown in the table below. Compared with C group, E group observed myocardial edema and decreased microvessel density. There was no significant difference between MHD₁₅ and MHD_15+E group. <h3>Conclusion</h3> Low dose irradiation of 15 Gy/3f (BED = 40 Gy) still cause myocardial fibrosis and damage of cardiac function in SD rats. RIHD were observed after 6 months, which was equal to about 1/5 of the rat's life span, and the time was delayed when compared with the time of RIHD caused by high dose irradiation. Cardiotoxicity was not increased significantly combining recombinant human endostatin on the basis of low-dose cardiac.

EGR1-CCL2 Feedback Loop Maintains Epithelial-Mesenchymal Transition of Cisplatin-Resistant Gastric Cancer Cells and Promotes Tumor Angiogenesis.

The mechanism of cisplatin resistance in gastric cancer (GC) is still elusive; several recent evidences proposed that chemoresistant tumor cells acquired aggressive behaviors. This study was aimed to investigate the mechanism of epithelial-mesenchymal transition (EMT) and angiogenesis in chemoresistant GC. Bioinformatics analysis and function or mechanism experiments including RT-qPCR, immunofluorescence, Western blot, luciferase reporter assay, Chromatin immunoprecipitation, Chicken chorioallantoic membrane assay and animal experiments were applied to evaluate the role of EGR1-CCL2 feedback loop. Compared with the parental cell line SGC7901, cisplatin resistant SGC7901R cells underwent EMT and showed increased angiogenic capabilities. Mechanistically, SGC7901R cells showed increased levels of EGR1, which could transcriptionally activate the angiogenic factor CCL2 and EMT regulator ZEB2. Reciprocally, CCL2 activated the CCR2-ERK-ELK1-EGR1 pathway, thus forming a positive feed-forward loop. Moreover, CCL2 in culture medium of SGC7901R cells promoted angiogenesis of Human Umbilical Vein Endothelial Cells (HUVECs). EGR1 expression was positively correlated with CCL2 and ZEB2 in clinical GC tissues, and the depletion of ERG1 could also decrease microvessel density and ZEB2 expression in metastatic nodules of nude mice. EGR1-CCL2 feedback loop might exert critical roles on EMT and angiogenesis of chemoresistant GC.

Effect of the Notch4/Dll4 signaling pathway in early gestational intrauterine infection on lung development.

Intrauterine infection is an important risk factor for bronchopulmonary dysplasia (BPD). BPD is characterized by arrested lung alveolarization and impaired pulmonary vascularization. The Notch4 signaling pathway is a key regulator of vascular remodeling and angiogenesis. Therefore, the presents study investigated the expression of Notch4, delta-like canonical Notch ligand 4 (Dll4) and related factors in an in vivo rat model and in rat pulmonary microvascular endothelial cells (PMVECs) in vitro, to study the mechanisms by which intrauterine infection affects rat lung development. A rat model of intrauterine infection was established by endocervical inoculation with Escherichia scoli on embryonic day 15. The date of birth was counted as postnatal day 0 (P0). Then, the lung tissues were collected from pups at days P3-P14. The expression of Notch4, Dll4 and related factors was measured by reverse transcription-quantitative PCR and western blotting. In addition, the γ-secretase inhibitor DAPT was used to examine the effect of Notch4 signaling on PMVECs. Intrauterine E. coli infection impaired normal lung development, as indicated by decreased microvessel density, fewer alveoli, fewer secondary septa, and larger alveoli compared with the control group. Furthermore, Notch4, Dll4 and NF-κB levels were significantly increased in the E. coli-infected group at P3 compared with the control group. Similarly, the mRNA expression levels of fetal liver kinase 1 (Flk-1, a VEGF receptor) were significantly increased in the E. coli-infected group at P3 and P7. In PMVECs, the inhibition of Notch4 signaling contributed to decreases in lipopolysaccharide (LPS)-induced expression of VEGF and its receptors. Furthermore, the inhibition of Notch4/Dll4 signaling accelerated cell proliferation and decreased the apoptosis rate of LPS-induced PMVECs. LPS-induced NF-κB expression in PMVECs was also attenuated by the Notch4/Dll4 inhibitor. In conclusion, intrauterine E. coli infection impaired normal lung development, possibly through Notch4/Dll4 signaling and effects on VEGF and its receptors.

Association of vaping with decreased vascular endothelial growth factor expression and decreased microvessel density in cutaneous wound healing tissue in rats.

Vaping is suggested to be a risk factor for poor wound healing akin to smoking. However, the molecular and histologic mechanisms underlying this postulation remain unknown. Our study sought to compare molecular and histologic changes in cutaneous flap and non-flap tissue between vaping, smoking and control cohorts. Animal study of 15 male Sprague-Dawley rats was randomized to three cohorts: negative control (n=5), e-cigarette (n=5) and cigarette (n=5) and exposed to their respective treatments with serum cotinine monitoring. After 30 days, random pattern flaps were raised and healed for 2 weeks after which skin punch biopsies of flap and non-flap tissues were collected for quantitative-reverse transcription-polymerase chain reaction of three selected wound healing genes (transforming growth factor β [TGF-β], vascular endothelial growth factor [VEGF], matrix metalloproteinase-1 [MMP-1]); then, immunohistochemistry for CD68 expression, α-smooth muscle actin looking at microvessel density (MVD) and in situ hybridization to localize VEGF production were undertaken. In flap tissue, vaping (mean[SEM]) (0.61[0.07]) and smoking (0.70[0.04]) were associated with decreased fold change of VEGF expression compared with controls (0.91[0.03]) (p < 0.05, p < 0.05, respectively). In non-flap tissue, only vaping was associated with decreased VEGF expression (mean[SEM]) (0.81[0.07]), compared with controls (1.17[0.10]) (p < 0.05) with expression primarily localized to basal keratinocytes and dermal capillaries. Immunohistochemistry showed decreased MVD in smoking (0.27[0.06]) and vaping (0.26[0.04]) flap tissue compared to matched controls (0.65[0.14]) (p < 0.05, p < 0.05, respectively) and decreased areas of fibrosis compared with controls on gross histology. Vaping and smoking were similarly associated with decreased VEGF expression, MVD and fibrotic changes in flap tissue. The results suggest attenuated angiogenesis via decreased VEGF expression as a mechanism for poor wound healing in vaping-exposed rats.

Alginic acid inhibits non-small cell lung cancer-induced angiogenesis via activating miR-506 expression.

Angiogenesis is a key event in non-small cell lung cancer progression. Alginic acid (AA), a kind of naturally occurring polyuronic acid, is generally enriched in edible brown algae. Recent studies have uncovered its anti-anaphylactic and anti-inflammatory properties. However, the effects of AA on human malignancies remain unknown. Herein, efficient inhibition of AA on NSCLC-induced angiogenesis was observed with tube formation and xenograft models. Subsequent results indicated that AA downregulated the expression of VEGF-A, a key angiogenesis-inducing cytokine. In addition, AA downregulated STAT3, a transcriptional inducer of VEGF-A and increased non-coding RNA miR-506 expression, respectively. Furthermore, miR-506 directly modulated STAT3 relying on base pairing the 3'-UTR in STAT3 mRNA. We also found that abrogation of miR-506 abolished the inhibitory effect of AA on VEGF-A expression and NSCLC-induced angiogenesis. Finally, xenografts experiments also showed that oral administration of AA could significantly attenuate NSCLC angiogenesis, indicated by decreased micro-vessel density (MVD) and the MVD marker CD31 expression in xenografts tissues. Correspondingly, AA treatment also downregulated VEGF-A, STAT3 and increased miR-506 expression in xenografts samples, respectively. Taken together, these results suggested that AA could suppress NSCLC-induced angiogenesis via miR-506/STAT3/VEGF-A axis. .

Discovery of novel N-benzylbenzamide derivatives as tubulin polymerization inhibitors with potent antitumor activities.

A series of novel N-benzylbenzamide derivatives were designed and synthesized as tubulin polymerization inhibitors. Among fifty-one target compounds, compound 20b exhibited significant antiproliferative activities with IC50 values ranging from 12 to 27nM against several cancer cell lines, and possessed good plasma stability and satisfactory physicochemical properties. Mechanism studies demonstrated that 20b bound to the colchicine binding site and displayed potent anti-vascular activity. Notably, the corresponding disodium phosphate 20b-P exhibited an excellent safety profile with the LD50 value of 599.7mg/kg (i.v. injection), meanwhile, it significantly inhibited tumor growth and decreased microvessel density in liver cancer cell H22 allograft mouse model without obvious toxicity. Collectively, 20b and 20b-P are novel promising anti-tubulin agents with more druggable properties and deserve to be further investigated for cancer therapy.

Cabozantinib can block growth of neuroendocrine prostate cancer patient-derived xenografts by disrupting tumor vasculature.

With the advent of potent second-line anti-androgen therapy, we and others have observed an increased incidence of androgen receptor (AR)-null small cell or neuroendocrine prostate cancer (SCNPC) in metastatic castration-resistant prostate cancer (mCRPC). Our study was designed to determine the effect of cabozantinib, a multi-targeted tyrosine kinase inhibitor that inhibits VEGFR2, MET and RET on SCNPC. Transcriptome analysis of the University of Washington rapid autopsy and SU2C mCRPC datasets revealed upregulated MET and RET expression in SCNPCs relative to adenocarcinomas. Additionally, increased MET expression correlated with attenuated AR expression and activity. In vitro treatment of SCNPC patient-derived xenograft (PDX) cells with the MET inhibitor AMG-337 had no impact on cell viability in LuCaP 93 (MET+/RET+) and LuCaP 173.1 (MET-/RET-), whereas cabozantinib decreased cell viability of LuCaP 93, but not LuCaP 173.1. Notably, MET+/RET+ LuCaP 93 and MET-/RET- LuCaP 173.1 tumor volumes were significantly decreased with cabozantinib treatment in vivo, and this activity was independent of MET or RET expression in LuCaP 173.1. Tissue analysis indicated that cabozantinib did not inhibit tumor cell proliferation (Ki67), but significantly decreased microvessel density (CD31) and increased hypoxic stress and glycolysis (HK2) in LuCaP 93 and LuCaP 173.1 tumors. RNA-Seq and gene set enrichment analysis revealed that hypoxia and glycolysis pathways were increased in cabozantinib-treated tumors relative to control tumors. Our data suggest that the most likely mechanism of cabozantinib-mediated tumor growth suppression in SCNPC PDX models is through disruption of the tumor vasculature. Thus, cabozantinib may represent a potential therapy for patients with metastatic disease in tumor phenotypes that have a significant dependence on the tumor vasculature for survival and proliferation.

Active metronomic vinorelbine schedules decrease plasma interleukin-2 levels in mice with Lewis lung carcinoma

The aim of our study was to investigate the effects of metronomic vinorelbine (mVNR) in a tumor model of Lewis Lung (LL) cancer in immunocompetent C57BL/6 mice, looking at the plasma levels of interleukin-2 (IL-2) and interleukin-8 (IL-8). mVNR caused a concentration-dependent antiproliferative effect in vitro on LL/2 cells. The in vivo experiment showed the significant antitumor effects of mVNR at the dose of 4 mg/Kg and 5 mg/Kg, 3 times/week, and the significant dose-dependent decrease of IL-2 concentrations in plasma samples. Conversely, such an effect was not observed for IL-8. A significant decrease in microvessel density was also found at both the active mVNR doses. In conclusion, our study confirmed the activity of mVNR in an immunocompetent model of lung carcinoma and suggest multiple mechanisms of action, including the modulation of IL-2 levels.