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

BACKGROUND: Excessive 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. METHODS: CRC 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. RESULTS: The 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. CONCLUSIONS: These 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.

Monitoring Bevacizumab-Induced Tumor Vascular Normalization by Intravoxel Incoherent Motion Diffusion-Weighted MRI.

BACKGROUND: Accurate monitoring of tumor blood vessel normalization progression is beneficial to accurate treatment of patients. At present, there is a lack of safe and noninvasive monitoring methods. PURPOSE: To serial monitor the vascular normalization time window of tumor antiangiogenesis treatment through intravoxel incoherent motion diffusion-weighted imaging (IVIM-DWI) and histopathological methods. STUDY TYPE: Exploratory animal study. POPULATION: Sixty rat C6 glioma models were randomly and equally divided into the control groups (N = 30) and bevacizumab treatment groups (N = 30). Twenty-five for magnetic resonance imaging (MRI) and five for electron microscope testing in each group. FIELD STRENGTH/SEQUENCE: T1-weighted imaging (T1WI), T2WI with a fast spin echo sequence and IVIM-DWI with a spin-echo echo-planar imaging sequence at 3 T. ASSESSMENT: IVIM-DWI quantitative parameters (f, D, D*, and fD*) were obtained on days 0, 2, 4, 6, and 8 after bevacizumab treatment. After MRI, the microvessel density (MVD), pericyte coverage, and hypoxia-inducible factor-1α (HIF-1α) were assessed. Electron microscope observation was performed at each time point. STATISTICAL TESTS: One-way analysis of variance and Student's t-tests were used to compare differences within and between groups. Spearman's correlation coefficient (r) assess the correlation between IVIM and pathological parameters. The intragroup correlation coefficient was determined to assess the repeatability of each IVIM parameter. RESULTS: The IVIM-DWI perfusion parameters (f and fD*) of the treated group were higher than the control group on days 2 and 4. Compared to the control group, MVD decreased on days 2 and pericyte coverage increased on days 4 in the treatment group. Electron microscopy showed that the tight junctions of the treatment group were prolonged on days 2-4. In the control group, f had the highest correlation with MVD (r = 0.689). In the treated group, f had a good correlation with pericyte coverage (r = 0.557), HIF-1α had a moderately positive correlation with f (r = 0.480) and fD*(r = 0.447). DATA CONCLUSION: The vascular normalization time window of bevacizumab treatment of glioma was days 2-4 after antiangiogenesis treatment, which could be monitored noninvasively by IVIM-DWI. EVIDENCE LEVEL: 2 TECHNICAL EFFICACY: Stage 3.

Pericyte hypoxia-inducible factor-1 (HIF-1) drives blood-brain barrier disruption and impacts acute ischemic stroke outcome.

Pericytes play essential roles in blood-brain barrier integrity and their dysfunction is implicated in neurological disorders such as stroke although the underlying mechanisms remain unknown. Hypoxia-inducible factor-1 (HIF-1), a master regulator of injury responses, has divergent roles in different cells especially during stress scenarios. On one hand HIF-1 is neuroprotective but on the other it induces vascular permeability. Since pericytes are critical for barrier stability, we asked if pericyte HIF-1 signaling impacts barrier integrity and injury severity in a mouse model of ischemic stroke. We show that pericyte HIF-1 loss of function (LoF) diminishes ischemic damage and barrier permeability at 3 days reperfusion. HIF-1 deficiency preserved barrier integrity by reducing pericyte death thereby maintaining vessel coverage and junctional protein organization, and suppressing vascular remodeling. Importantly, considerable improvements in sensorimotor function were observed in HIF-1 LoF mice indicating that better vascular functionality post stroke improves outcome. Thus, boosting vascular integrity by inhibiting pericytic HIF-1 activation and/or increasing pericyte survival may be a lucrative option to accelerate recovery after severe brain injury.

Ablation of endothelial prolyl hydroxylase domain protein-2 promotes renal vascular remodelling and fibrosis in mice.

Accumulating evidence demonstrates that hypoxia-inducible factor (HIF-α) hydroxylase system has a critical role in vascular remodelling. Using an endothelial-specific prolyl hydroxylase domain protein-2 (PHD2) knockout (PHD2EC KO) mouse model, this study investigates the regulatory role of endothelial HIF-α hydroxylase system in the development of renal fibrosis. Knockout of PHD2 in EC up-regulated the expression of HIF-1α and HIF-2α, resulting in a significant decline of renal function as evidenced by elevated levels of serum creatinine. Deletion of PHD2 increased the expression of Notch3 and transforming growth factor (TGF-ß1) in EC, thus further causing glomerular arteriolar remodelling with an increased pericyte and pericyte coverage. This was accompanied by a significant elevation of renal resistive index (RI). Moreover, knockout of PHD2 in EC up-regulated the expression of fibroblast-specific protein-1 (FSP-1) and increased interstitial fibrosis in the kidney. These alterations were strongly associated with up-regulation of Notch3 and TGF-ß1. We concluded that the expression of PHD2 in endothelial cells plays a critical role in renal fibrosis and vascular remodelling in adult mice. Furthermore, these changes were strongly associated with up-regulation of Notch3/TGF-ß1 signalling and excessive pericyte coverage.

Vascular abnormalities and development of hypoxia in microscopic melanoma xenografts.

BACKGROUND: Studies investigating the oxygenation status and the development of hypoxia in microscopic tumors are sparse. The purpose of this study was to measure the extent of hypoxia in microscopic melanoma xenografts and to search for possible mechanisms leading to the development of hypoxia in these tumors. METHODS: A-07, D-12, R-18, and U-25 human melanoma xenografts grown in dorsal window chambers or as flank tumors were used as preclinical tumor models. Morphologic and functional parameters of vascular networks were assessed with intravital microscopy, and the expression of angiogenesis-related genes was assessed with quantitative PCR. Microvessels, pericytes, and the extent of hypoxia were assessed by immunohistochemistry in microscopic tumors by using CD31, αSMA, and pimonidazole as markers, and the extent of radiobiological hypoxia was assessed in macroscopic flank tumors. RESULTS: Macroscopic R-18 and U-25 tumors showed extensive hypoxia, whereas macroscopic A-07 and D-12 tumors were less hypoxic. R-18 and U-25 tumors developed hypoxic regions before they reached a size of 2-3 mm in diameter, whereas A-07 and D-12 tumors of similar size did not show hypoxic regions. The development of hypoxic regions was not caused by low vessel density, but was rather a result of inadequate vascular function. Inadequate vascular function was not caused by low vessel diameters or long vessel segments, but was associated with poor vascular pericyte coverage. Poor pericyte coverage was associated with the expression of eight angiogenesis-related genes. CONCLUSIONS: Two of the four investigated melanoma models developed hypoxic regions in microscopic tumors, and the development of hypoxia was associated with poor vascular pericyte coverage and inadequate vascular function.

Pericyte loss leads to microvessel remodeling and nasal polyp formation.

BACKGROUND: Chronic rhinosinusitis (CRS) may be caused by increased vascular permeability and inflammatory cell leakage in the subepithelial tissue. AIMS/OBJECTIVES: The aim of this study is to clarify the role of pericytes in tissue edema, microvessel dysfunction and vascular remodeling mechanisms in patients of CRS with nasal polyps (CRSwNP). MATERIAL AND METHODS: A total of 63 tissue samples were collected, including 42 CRSwNP samples (22 eosinophilic CRSwNP (eCRSwNP) and 20 non-eosinophilic CRSwNP (non-eCRSwNP) samples) and 21 samples of CRS without nasal polyps (CRSsNP). The samples were stained by immunofluorescence to measure microvessel density (MVD) and microvessel pericyte coverage index (MPI). RESULTS: We found that the albumin expression in the eCRSwNP group was significantly increased (p < .05). The MPI was significantly decreased (p <.05). There was a significant negative correlation between the MPI and the plasma albumin level (r=-0.82, p < .05). The MPI was negatively correlated with eosinophilic count (r=-0.77, p < .05). In the eCRSwNP group, the expressions of IL-4, Ang-1 and Ang-2 were increased compared with those in the control group. CONCLUSIONS AND SIGNIFICANCE: Pericyte loss may induce microvessel dysfunction, affect the development of interstitial edema and eosinophilic exosmosis in eCRSwNP, and contribute to the formation and maintenance of nasal polyps.

Targeting OLFML3 in Colorectal Cancer Suppresses Tumor Growth and Angiogenesis, and Increases the Efficacy of Anti-PD1 Based Immunotherapy.

The role of the proangiogenic factor olfactomedin-like 3 (OLFML3) in cancer is unclear. To characterize OLFML3 expression in human cancer and its role during tumor development, we undertook tissue expression studies, gene expression analyses of patient tumor samples, in vivo studies in mouse cancer models, and in vitro coculture experiments. OLFML3 was expressed at high levels, mainly in blood vessels, in multiple human cancers. We focused on colorectal cancer (CRC), as elevated expression of OLFML3 mRNA correlated with shorter relapse-free survival, higher tumor grade, and angiogenic microsatellite stable consensus molecular subtype 4 (CMS4). Treatment of multiple in vivo tumor models with OLFML3-blocking antibodies and deletion of the Olfml3 gene from mice decreased lymphangiogenesis, pericyte coverage, and tumor growth. Antibody-mediated blockade of OLFML3 and deletion of host Olfml3 decreased the recruitment of tumor-promoting tumor-associated macrophages and increased infiltration of the tumor microenvironment by NKT cells. Importantly, targeting OLFML3 increased the antitumor efficacy of anti-PD-1 checkpoint inhibitor therapy. Taken together, the results demonstrate that OLFML3 is a promising candidate therapeutic target for CRC.

Corrigendum: Changes in Tumor-Infiltrating Lymphocytes and Vascular Normalization in Breast Cancer Patients After Neoadjuvant Chemotherapy and Their Correlations With DFS.

[This corrects the article DOI: 10.3389/fonc.2019.01545.].

Thrombin promotes pericyte coverage by Tie2 activation in a rat model of intracerebral hemorrhage.

Pericyte coverage on the endothelial tubes leads to the formation of a mature and stable microvessel system, which is critical for brain repair after intracerebral hemorrhage (ICH). We report herein that thrombin promotes pericyte coverage by activating Tie2 and the downstream signaling pathway PI3K/Akt in a rat model of ICH. ICH was induced by injection of autologous blood with or without thrombin inhibitor hirudin. Rats were treated with thrombin alone or in combination with a Tie2 inhibitor. The expression of total- and phospho-Tie2, PI3K and phospho-Akt, blood perfusion, pericyte coverage, IgG extravasation, neuron survival and neurological deficits were evaluated by western blot, fluorescein-5-isothiocyanate-dextran staining, immunohistochemistry, Nissl staining and modified neurological severity scores respectively. Induction of ICH resulted in increased phosphorylation of Tie2 on endothelial cells and pericyte coverage, better formation of integral and functional microvessels, more surviving neurons and accelerated motor function recovery, all of which were significantly attenuated by hirudin at 7 and 14 days after ICH induction. Furthermore, thrombin increased phosphorylation of Tie2 and Akt, expression of PI3K, and pericyte coverage, which were however reversed by pharmacological inhibition of Tie2. Our results demonstrated that thrombin promotes pericyte coverage on microvessels following ICH by enhancing activation of Tie2, in which the downstream PI3K/Akt signaling pathway might be involved.

Changes in Tumor-Infiltrating Lymphocytes and Vascular Normalization in Breast Cancer Patients After Neoadjuvant Chemotherapy and Their Correlations With DFS.

Objective: Changes in the number of various tumor-infiltrating lymphocytes (TILs) and degrees of vascular normalization in breast cancer (BC) patients after neoadjuvant chemotherapy (NAC) were analyzed to screen key factors that can predict the prognosis. Methods: HE-stained sections were used to assess the degree of TILs infiltration; immunohistochemically stained sections were used to assess the infiltration of CD8+, CD4+, FOXP3+ Tregs and the expression of PD-L1; immunofluorescence-stained sections were used to assess the microvessel density (MVD) and microvessel pericyte coverage index (MPI). The expression of them before NAC were compared with those after NAC, and correlations between changes in these parameters and the pathological complete remission (pCR) and DFS of BC patients were analyzed. Results: After NAC, the percentage of patients with enhanced sTILs in the pCR group was significantly higher than that in the Non-pCR group (P < 0.05). Univariate and multivariate analyses showed that the number of FOXP3+ Tregs and MPI before NAC were correlated with pCR (P < 0.05). Survival analysis showed that the DFS of BC patients with reduced FOXP3+ Tregs was significantly better than that of patients with elevated FOXP3+ Tregs (P = 0.029). The sTILs count and MPI were significantly higher in primary tumors than lymph nodes (P < 0.05). Conclusion: After NAC, the reduced infiltration of FOXP3+ Tregs was correlated with an improvement in DFS in BC patients. Changes in the number of FOXP3+ Tregs and the MPI may be used as prognostic markers for BC patients.

Cerebral microvascular abnormalities in patients with idiopathic intracranial hypertension.

AIM: Idiopathic intracranial hypertension (IIH) is characterized by symptoms indicative of increased intracranial pressure (ICP), such as headache and visual impairment. We have previously reported that brain biopsies from IIH patients show patchy astrogliosis and increased expression of the water channel aquaporin-4 (AQP4) at perivascular astrocytic endfeet. METHODS: The present study was undertaken to investigate for ultrastructural changes of the cerebral capillaries in individuals with IIH. We examined by electron microscopy (EM) biopsies from the cortical parenchyma of 10 IIH patients and 8 reference subjects (patients, not healthy individuals), in whom tissue was retrieved from other elective and necessary brain surgeries (epilepsy, tumors or vascular diseases). IIH patients were diagnosed on the basis of typical clinical symptoms and abnormal intracranial pressure wave amplitudes during overnight ICP monitoring. RESULTS: All 10 IIH patients underwent shunt surgery followed by favorable clinical outcome. EM revealed abnormal pericyte processes in IIH. The basement membrane (BM) showed more frequently evidence of degeneration in IIH, but neither the BM dimensions nor the pericyte coverage differed between IIH and reference tissue. The BM thickness increased significantly with increasing age. Reference individuals were older than IIH cases; observations may to some extent be age-related. CONCLUSION: The present study disclosed marked changes of the cerebral cortical capillaries in IIH patients, suggesting that microvascular alterations are involved in the evolvement of IIH.

Brain Capillary Ultrastructure in Idiopathic Normal Pressure Hydrocephalus: Relationship With Static and Pulsatile Intracranial Pressure.

Idiopathic normal pressure hydrocephalus (iNPH) is a neurodegenerative disease of unknown cause. We investigated the morphology of capillaries in frontal cortex biopsies from iNPH patients and related the observations to overnight intracranial pressure (ICP) scores. A biopsy (0.9×10 mm) was taken from where the ICP sensor subsequently was inserted. Brain capillaries were investigated by electron microscopy of biopsies from 27 iNPH patients and 10 reference subjects, i.e. patients (not healthy individuals) without cerebrospinal fluid circulation disturbances, in whom normal brain tissue was removed as part of necessary neurosurgical treatment. Degenerating and degenerated pericyte processes were identified in 23/27 (85%) iNPH and 6/10 (60%) of reference specimens. Extensive disintegration of pericyte processes were recognized in 11/27 (41%) iNPH and 1/10 (10%) reference specimens. There were no differences in basement membrane (BM) thickness or pericyte coverage between iNPH and reference subjects. The pulsatile or static ICP scores did neither correlate with the BM thickness nor with pericyte coverage. We found increased prevalence of degenerating pericytes in iNPH while the BM thickness and pericyte coverage did not differ from the reference individuals. Observations in iNPH may to some extent be age-related since the iNPH patients were significantly older than the reference individuals.

Tumor Angiogenesis in Breast Cancer: Pericytes and Maturation Does Not Correlate With Lymph Node Metastasis and Molecular Subtypes.

BACKGROUND: Angiogenesis, traditionally assessed by microvessel density (MVD), does not give an indication of the functional status of the tumor neovasculature. The structural and functional stability of the tumor vasculature and its potential clinical relevance in breast cancer was evaluated. MATERIALS AND METHODS: In invasive breast cancer, immunostaining of endothelial cells and pericytes was performed using anti-CD34 and anti-platelet-derived growth factor receptor-ß antibody, respectively. Double immunostaining for the proliferating capillary index (PCI) and microvessel pericyte coverage index (MPI) was performed with CD34/Ki-67 and CD34/smooth muscle actin. RESULTS: The mean MVD of 145 vessels/mm(2) was significantly greater in grade 3 tumors (P = .018) and in necrotic tumors (P = .022). The PCI ranged from 0% to 17.14% (mean, 4.37%) and was associated with a high proliferative index in tumor tissue (P = .044). The MPI ranged from 13.09% to 88.18% (mean, 41.35%), indicating the stability of the tumor vasculature. However, it was not significantly associated with the tumor size, tumor grade, lymph node metastasis, proliferative index, or molecular subtypes. CONCLUSION: MVD remains the angiogenesis-related parameter associated with tumor grade and necrosis. The PCI was the only functional parameter of angiogenesis associated with a poor prognostic indicator. The MPI did not show any correlation with the known prognostic and predictive factors.

Lenvatinib, an angiogenesis inhibitor targeting VEGFR/FGFR, shows broad antitumor activity in human tumor xenograft models associated with microvessel density and pericyte coverage.

BACKGROUND: Lenvatinib is an oral inhibitor of multiple receptor tyrosine kinases (RTKs) targeting vascular endothelial growth factor receptor (VEGFR1-3), fibroblast growth factor receptor (FGFR1-4), platelet growth factor receptor α (PDGFR α), RET and KIT. Antiangiogenesis activity of lenvatinib in VEGF- and FGF-driven angiogenesis models in both in vitro and in vivo was determined. Roles of tumor vasculature (microvessel density (MVD) and pericyte coverage) as biomarkers for lenvatinib were also examined in this study. METHOD: We evaluated antiangiogenesis activity of lenvatinib against VEGF- and FGF-driven proliferation and tube formation of HUVECs in vitro. Effects of lenvatinib on in vivo angiogenesis, which was enhanced by overexpressed VEGF or FGF in human pancreatic cancer KP-1 cells, were examined in the mouse dorsal air sac assay. We determined antitumor activity of lenvatinib in a broad panel of human tumor xenograft models to test if vascular score, which consisted of high MVD and low pericyte coverage, was associated with sensitivity to lenvatinib treatment. Vascular score was also analyzed using human tumor specimens with 18 different types of human primary tumors. RESULT: Lenvatinib inhibited VEGF- and FGF-driven proliferation and tube formation of HUVECs in vitro. In vivo angiogenesis induced by overexpressed VEGF (KP-1/VEGF transfectants) or FGF (KP-1/FGF transfectants) was significantly suppressed with oral treatments of lenvatinib. Lenvatinib showed significant antitumor activity in KP-1/VEGF and five 5 of 7 different types of human tumor xenograft models at between 1 to 100 mg/kg. We divided 19 human tumor xenograft models into lenvatinib-sensitive (tumor-shrinkage) and relatively resistant (slow-growth) subgroups based on sensitivity to lenvatinib treatments at 100 mg/kg. IHC analysis showed that vascular score was significantly higher in sensitive subgroup than relatively resistant subgroup (p < 0.0004). Among 18 types of human primary tumors, kidney cancer had the highest MVD, while liver cancer had the lowest pericyte coverage, and cancers in Kidney and Stomach had highest vascular score. CONCLUSION: These results indicated that Lenvatinib inhibited VEGF- and FGF-driven angiogenesis and showed a broad spectrum of antitumor activity with a wide therapeutic window. MVD and pericyte-coverage of tumor vasculature might be biomarkers and suggest cases that would respond for lenvatinib therapy.

Cytological study on rat corneal new blood vessel after alkali burn / 中华实验眼科杂志

Background Microvessels are composed of two interacting cell types: endothelial cells and pericytes. Over the past decades, studies of corneal angiogenesis have concentrated mainly on endothelial cells, while interest in pericytes has lagged behind. Objective The present study aimed to investigate the recruitment of vascular endothelial cells and pericytes in rat corneas after alkali burn. Methods Corneal alkali burn models were established in the right eyes of 36 adult SPF SD rats by putting 4 mm medicators containing a 1% 1 mol/L NaOH solution at the central corneas for 30 seconds, and 3 matched normal rats were used as controls. Corneas were excised 1,2,3,5,7 and 10 days after surgery. Frozen sections that parallel with the corneoscleral limbus were constructed. Double immunofluorescence staining was used to observe the dynamic expression of CD31 and α-smooth muscle actin (α-SMA) in corneal tissue for the evaluation of the number of endothelial cells and pericytes. The pericyte coverage index (PCI) was calculated to quantify the recruitment of pericytes to neovascular sites. The use of experimental animals followed the Statement of Association for Research in Vision and Ophthalmology. Results CD31 was expressed in the superficial stromal layer of the cornea on the 1 st day, showing the presence of red fluoresence. The positive cell number for CD31 was gradually increased with the passage of time and proceeded into the deep stromal layer from days 2 through 5 but decreased after that. However,α-SMA was positively expressed on the 2nd day in the cornea after alkali burn with the presence of green fluorescence. The positive cell number for α-SMA was less than those of CD31 throughout the experimental period. The PCI was 0, 16.07%, 11.95%, 43.84%, 73.97% and 86. 21% , respectively, 1,2,3,5,7 and 10 days after surgery. Conclusion Pericytes recruitment to corneal new vessels may play a key role in the stabilization and maturation of angiogeneis.