Innovative assessment of lipid-induced oxidative stress and inflammation in harvested human endothelial cells.

Studying acute changes in vascular endothelial cells in humans is challenging. We studied ten African American women and used the J-wire technique to isolate vein endothelial cells before and after a four-hour lipid and heparin infusion. Dynamic changes in lipid-induced oxidative stress and inflammatory markers were measured with fluorescence-activated cell sorting. We used the surface markers CD31 and CD144 to identify human endothelial cells. Peripheral blood mononuclear cells isolated from blood were used as a negative control. The participants received galantamine (16 mg/day) for 3 months. We previously demonstrated that galantamine treatment effectively suppresses lipid-induced oxidative stress and inflammation. In this study, we infused lipids to evaluate its potential to increase the activation of endothelial cells, as assessed by the levels of CD54+ endothelial cells and expression of Growth arrest-specific 6 compared to the baseline sample. Further, we aimed to investigate whether lipid infusion led to increased expression of the oxidative stress markers IsoLGs and nitrotyrosine in endothelial cells. This approach will expedite the in vivo identification of novel pathways linked with endothelial cell dysfunction induced by oxidative stress and inflammatory cytokines. This study describes an innovative method to harvest and study human endothelial cells and demonstrates the dynamic changes in oxidative stress and inflammatory markers release induced by lipid infusion.

Utility of Platelet Endothelial Cell Adhesion Molecule 1 in the Platelet Activity Assessment in Mouse and Human Blood.

In our previous study, we introduced the platelet endothelial cell adhesion molecule 1 (PECAM-1)/thrombus ratio, which is a parameter indicating the proportion of PECAM-1 in laser-induced thrombi in mice. Because PECAM-1 is an antithrombotic molecule, the higher the PECAM-1/thrombus ratio, the less activated the platelets. In this study, we used an extracorporeal model of thrombosis (flow chamber model) to verify its usefulness in the assessment of the PECAM-1/thrombus ratio in animal and human studies. Using the lipopolysaccharide (LPS)-induced inflammation model, we also evaluated whether the PECAM-1/thrombus ratio determined in the flow chamber (without endothelium) differed from that calculated in laser-induced thrombosis (with endothelium). We observed that acetylsalicylic acid (ASA) decreased the area of the thrombus while increasing the PECAM-1/thrombus ratio in healthy mice and humans in a dose-dependent manner. In LPS-treated mice, the PECAM-1/thrombus ratio decreased as the dose of ASA increased in both thrombosis models, but the direction of change in the thrombus area was inconsistent. Our study demonstrates that the PECAM-1/thrombus ratio can more accurately describe the platelet activation status than commonly used parameters such as the thrombus area, and, hence, it can be used in both human and animal studies.

In vitro and in vivo assessment of controlled release and degradation of acoustically responsive scaffolds.

Spatiotemporally controlled release of growth factors (GFs) is critical for regenerative processes such as angiogenesis. A common strategy is to encapsulate the GF within hydrogels, with release being controlled via diffusion and/or gel degradation (i.e., hydrolysis and/or proteolysis). However, simple encapsulation strategies do not provide spatial or temporal control of GF delivery, especially non-invasive, on-demand controlled release post implantation. We previously demonstrated that fibrin hydrogels, which are widely used in tissue engineering and GF delivery applications, can be doped with perfluorocarbon emulsion, thus yielding an acoustically responsive scaffold (ARS) that can be modulated with focused ultrasound, specifically via a mechanism termed acoustic droplet vaporization. This study investigates the impact of ARS and ultrasound properties on controlled release of a surrogate payload (i.e., fluorescently-labeled dextran) and fibrin degradation in vitro and in vivo. Ultrasound exposure (2.5MHz, peak rarefactional pressure: 8MPa, spatial peak time average intensity: 86.4mW/cm2), generated up to 7.7 and 21.7-fold increases in dextran release from the ARSs in vitro and in vivo, respectively. Ultrasound also induced morphological changes in the ARS. Surprisingly, up to 2.9-fold greater blood vessel density was observed in ARSs compared to fibrin when implanted subcutaneously, even without delivery of pro-angiogenic GFs. The results demonstrate the potential utility of ARSs in generating controlled release for tissue regeneration. STATEMENT OF SIGNIFICANCE: Simple encapsulation of a molecular payload within a conventional hydrogel scaffold does not provide spatial or temporal control of payload release. Yet, spatiotemporally controlled release of bioactive payloads is critical for tissue regeneration, which often utilizes hydrogel scaffolds to facilitate processes such as angiogenesis. This work investigates the design and performance (both in vitro and in vivo) of hydrogel scaffolds where release of a fluorescent payload is non-invasively and spatiotemporally-controlled using focused ultrasound. We also quantitatively characterize the degradation and vascularization of the scaffolds. Our results may be of interest to groups working on controlled release strategies for implants, especially within the field of tissue engineering.

Bioprinted 3D Primary Liver Tissues Allow Assessment of Organ-Level Response to Clinical Drug Induced Toxicity In Vitro.

Modeling clinically relevant tissue responses using cell models poses a significant challenge for drug development, in particular for drug induced liver injury (DILI). This is mainly because existing liver models lack longevity and tissue-level complexity which limits their utility in predictive toxicology. In this study, we established and characterized novel bioprinted human liver tissue mimetics comprised of patient-derived hepatocytes and non-parenchymal cells in a defined architecture. Scaffold-free assembly of different cell types in an in vivo-relevant architecture allowed for histologic analysis that revealed distinct intercellular hepatocyte junctions, CD31+ endothelial networks, and desmin positive, smooth muscle actin negative quiescent stellates. Unlike what was seen in 2D hepatocyte cultures, the tissues maintained levels of ATP, Albumin as well as expression and drug-induced enzyme activity of Cytochrome P450s over 4 weeks in culture. To assess the ability of the 3D liver cultures to model tissue-level DILI, dose responses of Trovafloxacin, a drug whose hepatotoxic potential could not be assessed by standard pre-clinical models, were compared to the structurally related non-toxic drug Levofloxacin. Trovafloxacin induced significant, dose-dependent toxicity at clinically relevant doses (≤ 4uM). Interestingly, Trovafloxacin toxicity was observed without lipopolysaccharide stimulation and in the absence of resident macrophages in contrast to earlier reports. Together, these results demonstrate that 3D bioprinted liver tissues can both effectively model DILI and distinguish between highly related compounds with differential profile. Thus, the combination of patient-derived primary cells with bioprinting technology here for the first time demonstrates superior performance in terms of mimicking human drug response in a known target organ at the tissue level.

Differentiation of functional endothelial cells from human induced pluripotent stem cells: A novel, highly efficient and cost effective method.

Endothelial cells derived from human induced pluripotent stem cells (hiPSC- EC) are of significant value for research on human vascular development, in vitro disease models and drug screening. Here we report an alternative, highly efficient and cost-effective simple three step method (mesoderm induction, endothelial cell differentiation and endothelial cell expansion) to differentiate hiPSC directly into endothelial cells. We demonstrate that efficiency of described method to derive CD31+ and VE-Cadherin+ double positive cells is higher than 80% in 12 days. Most notably we established that hiPSC-EC differentiation efficacy depends on optimization of both mesoderm differentiation and endothelial cell differentiation steps.

Gd-loaded-RBCs for the assessment of tumor vascular volume by contrast-enhanced-MRI.

The assessment of the fractional vascular volume (vV) in the tumor area is of great interest in the characterization of tumor and it can be useful to monitor the outcome of anti-angiogenetic therapies. The high spatial and temporal resolution of Magnetic Resonance Imaging makes it the election imaging modality to monitor in vivo the vascular volume changes. Commonly used MRI methods to obtain this information rely on the administration of contrast agents that modify the bulk water relaxation times but, unfortunately, they can provide only an estimate of vV since they are not fully retained in the vascular space. Herein, Gd-loaded Red Blood Cells (Gd-RBCs) are proposed as a contrast agent able to provide quantitative information on tumor vascularization. Being Gd-RBCs fully retained in the vascular space, the proposed method does not suffer for the limitations associated to the use of extracellular Gd-agents that quickly extravasate in the leaky tumor vasculature. Furthermore, the long half-life and biocompatibility of Gd-RBCs allows repeating the measurement many times upon their administration; this ensures the possibility to in vivo evaluate the change of vascular volume during tumor growth. For these reasons, Gd-RBCs may represent a highly biocompatible imaging reporter of vasculature, able to quantitatively assess changes in the vascular volume in the ROI.

Immunohistochemical assessment of lymphatic and blood vessel invasion in T1 urothelial carcinoma of the bladder.

OBJECTIVE: The aim of this study was evaluate the incidence and significance of immunohistochemically assessed lymphatic (LVI) and blood vessel invasion (BVI) in primary T1 urothelial carcinoma of the bladder (UCB) treated with radical cystectomy (RC). MATERIALS AND METHODS: Thirty-two patients with T1 UCB at primary diagnosis were identified who underwent radical cystectomy (RC) subsequently. Of these, 16 (50%) had pT1N0M0 (group I) and 16 (50%) ≥ pT2aN0-3M0 UCB (group II) at RC. The presence of LVI and BVI in transurethral resection of bladder tumor (TURBT) and corresponding RC specimens was assessed using hematoxylin & eosin (H&E) and immunohistochemical (IHC) staining against the lymphatic (D2-40) and vascular endothelium (CD31). RESULTS: At TURBT and RC, none of the patients in group I showed LVI or BVI on H&E and IHC sections. In group II, at TURBT, LVI and BVI were negative on H&E staining in all patients, but detectable by IHC in two patients (13%) and one patient (6%), respectively (p = 0.48 and p = 0.99 compared to group I). At RC, LVI and BVI were detected by IHC in eight (50%) and five (31%) of the 16 patients, respectively (p = 0.002 and p = 0.021 compared to group I). Of these eight and five patients, detection of LVI and BVI was only possible with IHC in six (75%) and three (60%), respectively. CONCLUSIONS: Although this hypothesis-generating study did not show a high degree of concordance between TURBT and RC specimens, IHC assessment on a regular basis may increase the detection rates of LVI and BVI at initial diagnosis and improve the selection of those T1 patients who should be offered early radical treatment.

Early assessment of tumor response to gefitinib treatment by noninvasive optical imaging of tumor vascular endothelial growth factor expression in animal models.

UNLABELLED: Epidermal growth factor receptor (EGFR) expression is upregulated in many types of tumors, and the EGFR tyrosine kinase inhibitor gefitinib has high potential as an anticancer drug. However, accumulating clinical evidence has indicated that only a subset of patients benefit from gefitinib treatment. This study aimed to determine whether optical imaging of vascular endothelial growth factor (VEGF) expression can be an early biomarker for tumor response to gefitinib therapy. METHODS: A VEGF-targeting fluorescent probe Dye-BevF(ab')2 was prepared and tested in vivo. Longitudinal optical imaging studies using Dye-BevF(ab')2 were performed in both 22B (gefitinib-resistant) and A549 (gefitinib-responsive) tumor models at different times (days 0, 2, and 5) before and after gefitinib treatment. The imaging results were validated by ex vivo immunofluorescence staining and enzyme-linked immunosorbent assay. RESULTS: Dye-BevF(ab')2 exhibited high specificity for VEGF in vivo. There was no significant change in the Dye-BevF(ab')2 uptake in gefitinib-treated 22B tumors, compared with the control group. In contrast, the A549 tumor uptake of Dye-BevF(ab')2 in the gefitinib-treated group was significantly lower on days 2 and 5 than that in the control group and at the baseline. An in vivo gefitinib treatment study confirmed that 22B tumors were gefitinib-resistant, whereas A549 tumors were gefitinib-responsive. Immunofluorescence staining and enzyme-linked immunosorbent assay confirmed that changes in the Dye-BevF(ab')2 uptake were correlated with VEGF expression levels in tumors. CONCLUSION: Optical imaging of VEGF expression with Dye-BevF(ab')2 can be used for the early assessment of tumor response to gefitinib therapy. This approach may also be valuable for preclinical high-throughput screening of novel antiangiogenic drugs.

Assessment of the prognostic significance of endoglin (CD105) in clear cell renal cell carcinoma using automated image analysis.

The behavior of clear cell renal cell carcinoma can be difficult to predict. Angiogenesis has proven to be a useful prognostic indicator in different malignancies. Endoglin (CD105) is a new marker of angiogenesis found to have prognostic utility in various tumors. Here, we provide the first automated digital assessment of intratumoral microvascular density in clear cell renal cell carcinoma using endoglin and CD31 and assess their utility as predictors of clinical outcome. Both endoglin and CD31 expression showed association with advanced tumor stage (P = .025 and P = .011, respectively). There was a significant correlation between CD31 and tumor grade (P = .034). Kaplan-Meier survival curves showed that patients with higher endoglin expression had significantly shorter progression-free survival (P = .010). Patients with higher CD31 expression tended to have a worse prognosis, although this was not statistically significant (P = .082). In univariate analysis using endoglin as a continuous variable, increased endoglin was strongly associated with reduced survival (hazard ratio, 1.74; 95% CI, 1.39-2.18; P = <.001). CD31 also correlated with poor outcomes (hazard ratio, 1.52; 95% CI, 1.24-1.86; P = .001). There was no correlation between CD31 and endoglin expression (r = -0.090, P = .541). Receiver operating characteristic analysis showed the area under the curve to be 0.749 for endoglin and 0.550 for CD31. In conclusion, increased endoglin and CD31 expression are associated with a higher tumor stage and decreased progression-free survival. Our automated approach overcomes many limitations of manual quantification. Advances in digital assessment of immunohistochemical markers can be helpful in standardizing the evaluation of tumor biomarkers.

Long-term assessment of prostate cancer progression free survival: evaluation of pathological parameters, nuclear shape and molecular biomarkers of pathogenesis.

BACKGROUND: Molecular pathways of proliferation, angiogenesis, neuroendocrine differentiation, apoptosis and alterations in nuclear structure of cancer epithelial cells are important in the pathogenesis of prostate cancer (PCa). Therefore, we evaluated the prognostic value of these parameters in 105 clinically localized PCa tumors with long-term follow-up after radical prostatectomy for progression-free survival (PFS). METHOD: Nuclear roundness variance (NRV) was calculated for tumor nuclei using the graphic tracing DynaCELL system. Immunohistochemistry assessed expression of Ki67, PCNA (proliferation), Chromogranin A (neuroendocrine differentiation), CD31 (angiogenesis), BCL2 (apoptosis), and Her-2/neu (oncogene) in the tumors. Cox proportional hazards regression, Spearman's rank correlation, and Kaplan-Meier plots were employed to analyze the data. RESULTS: Gleason score, focal vs. non-focal extra-prostatic extension, organ confined status, NRV, Her-2/neu, CD-31 and Ki67 were univariately significant predictors of PFS. NRV was the most significant prognostic indicator with the highest concordance index (0.7) for PFS. Gleason score, NRV and Her-2/neu were multivariately significant and yielded a concordance index of 0.77. CONCLUSION: Her-2/neu oncogene and NRV were shown to be significant in the prediction of PFS. The assessment of alterations in nuclear structure using NRV proved to be the most significant factor in the prediction of PFS. Integration of image analysis-based NRV and molecular biomarkers with pathologic parameters should be considered for validation in the prediction of PFS.

Assessment of alphavbeta3 integrin expression after myocardial infarction by positron emission tomography.

AIMS: The purpose of this study was to determine the feasibility of a new positron emission tomography (PET) imaging approach using an (18)F-labelled alpha(v)beta(3) integrin antagonist ((18)F-Galacto-RGD) to monitor the integrin expression after myocardial infarction. METHODS AND RESULTS: Male Wister rats were subjected to 20 min transient left coronary artery occlusion followed by reperfusion. Autoradiographic analysis and in vivo PET imaging were used to determine myocardial (18)F-Galacto-RGD uptake at different time points following reperfusion. RESULTS: PET imaging and autoradiography demonstrated no significant focal myocardial (18)F-Galacto-RGD uptake in non-operated control rats and at day 1 after reperfusion. However, focal accumulation in the infarct area started at day 3 (uptake ratio = 1.91 +/- 0.22 vs. remote myocardium), peaked between 1 (3.43 +/- 0.57) and 3 weeks (3.43 +/- 0.95), and decreased to 1.96 +/- 0.40 at 6 months after reperfusion. Pretreatment with alpha(v)beta(3) integrin antagonist c(-RGDfV-) significantly decreased tracer uptake, indicating the specificity of tracer uptake. The time course of focal tracer uptake paralleled vascular density as measured by CD31 immunohistochemical analysis. CONCLUSION: Regional (18)F-Galacto-RGD accumulation suggests up-regulation of alpha(v)beta(3) integrin expression after myocardial infarction, which peaks between 1 and 3 weeks and remains detectable until 6 months after reperfusion. This new PET tracer is promising for the monitoring of myocardial repair processes.

An indirect quantitative method for the assessment of angiogenesis in human tissues.

We set up an ELISA that measures the concentration of von Willebrand factor (vWF) in human endometrial tissue and found a significant correlation with the mean vessel counts in vWF- and CD31-immunostained tissue sections. This ELISA allows an objective and quantitative evaluation of the vascular state in the endometrium and could be used as a method to evaluate the angiogenic state in other tissues.

Assessment of vascular maturation in lung and breast carcinomas using a novel basement membrane component, LH39.

LH39, is a monoclonal antibody recognizing an epitope located at the lamina lucida of mature small veins and capillaries but not in newly- formed vessels of several pathological conditions including cancer. We examined the ratio of mature/immature vessels in 50 breast and 81 lung carcinomas and correlated the vascular maturation index (VMI) to different clinicopathological variables including angiogenesis. Mature vessels were defined by staining with antibodies to both LH39 and CD31, using double immunohistochemistry, whereas immature vessels stained only for CD31. VMI was defined as the percentage fraction of mature vessels (LH39 positive)/total number of vessels (CD31 positive). VMI in breast carcinomas ranged from 0-47% (median 8.75%), which was significantly lower than that observed in the normal breast cases (range 54%-70%; median 68%). The median VMI in the non-small cell lung carcinomas was 46% (range 15%-90%). There was a significant inverse correlation between high tumor VMI and absence of nodal involvement in both breast and lung tumors examined (p=0.01). Thymidine phosphorylase (TP) expression, but not vascular endothelial growth factor (VEGF) expression, was related to a low VMI showing an intense vascular remodeling in TP expressing cases. Thus, assessment of vessel maturation might be complementary to microvessel number to aid the identification of patients who might benefit from specific antiangiogenic therapies or vascular targeting treatment.