Neovascular Structures Research Articles

Treating ischemic stroke by improving vascular structure and promoting angiogenesis using Taohong Siwu Decoction: An integrative pharmacology strategy

Ethnopharmacological relevanceNeovessels represent a crucial therapeutic target and strategy for repairing ischemic tissue. Taohong Siwu Decoction (THSWD) exhibits potential in promoting angiogenesis to address ischemic stroke (IS). However, its impact on neovessel structure and function, alongside the underlying molecular mechanisms, remains elusive. Aim of the studyOur aim is to investigate the protective effects of THSWD on neovessel structure and function, as well as the associated molecular mechanisms, utilizing an integrative pharmacological approach. Materials and methodsWe initially employed behavioral tests, 2,3,5-triphenyltetrazolium chloride (TTC) staining, Haematoxylin-eosin (HE) staining, enzyme-linked immunosorbent assay (ELISA), Laser Doppler flowmetry (LDF), Evans blue staining, and immunofluorescence to evaluate the protective effects of THSWD on neovascular structure and function in middle cerebral artery occlusion/reperfusion (MCAO/R) rats. Subsequently, we utilized network pharmacology, metabolomics, and experimental validation to elucidate the underlying molecular mechanisms of THSWD in enhancing neovascular structure and function. ResultIn addition to significantly reducing neurological deficits and cerebral infarct volume, THSWD mitigated pathological damage, blood-brain barrier (BBB) leakage, and cerebral blood flow disruption. Moreover, it preserved neovascular structure and stimulated angiogenesis. THSWD demonstrated potential in ameliorating cerebral microvascular metabolic disturbances including lipoic acid metabolism, fructose and mannose metabolism, purine metabolism, and ether lipid metabolism. Consequently, it exhibited multifaceted therapeutic effects, encompassing anti-inflammatory, antioxidant, energy metabolism modulation, and antiplatelet aggregation properties. ConclusionTHSWD exhibited protective effects on cerebral vascular structure and function and facilitated angiogenesis by rectifying cerebral microvascular metabolic disturbances in MCAO/R rats. Furthermore, integrated pharmacology offers a promising approach for studying the intricate traditional Chinese medicine (TCM) system in IS treatment.

Assessing the fate and contribution of Foxd1-expressing embryonic precursors and their progeny in palatal development, homeostasis and excisional repair

Oral mucosal tissues heal rapidly with minimal scarring, although palatal mucosa can be associated with excessive fibrosis in response to injury. Investigations on the balance between neovascularization and tissue repair suggests regulation of angiogenesis is an important determinant of repair versus scarring. Associated with pericyte mediated fibrosis in kidney injury, FoxD1 is implicated in growth centres during cranio-facial development, although which cell lineages are derived from these embryonic populations in development and in adult animals is unknown. Using a lineage tracing approach, we assessed the fate of embryonic Foxd1-expressing progenitor cells and their progeny in palatal development and during wound healing in adult mice. During palatal development as well as in post-natal tissues, Foxd1-lineage progeny were associated with the vasculature and the epineurium. Post-injury, de novo expression of FoxD1 was not detectable, although Foxd1-lineage progeny expanded while exhibiting low association with the fibroblast/myofibroblast markers PDGFα, PDGFβ, vimentin, α-smooth muscle actin, as well as the neuronal associated markers S100β and p75NTR. Foxd1-lineage progeny were primarily associated with CD146, CD31, and to a lesser extent CD105, remaining in close proximity to developing neovascular structures. Our findings demonstrate that FoxD1 derived cells are predominantly associated with the palatal vasculature and provide strong evidence that FoxD1 derived cells do not give rise to populations involved directly in the scarring of the palate.

MODL-42. BLOOD-BRAIN TUMOR BARRIER ORGANOID MODEL FOR THERAPEUTIC DELIVERY TO GLIOBLASTOMA

Abstract The integrity of the blood-brain barrier (BBB) is compromised in brain tumors, leading to the formation of the blood-brain tumor barrier (BTB). While the BTB is generally more permeable than the BBB, it is nevertheless heterogeneously restrictive to the entry of therapeutic agents to brain tumors. Modeling the BTB accurately is crucial for understanding its physiological properties and developing effective treatments. Our lab has previously introduced a 3D in vitro human BBB organoid platform that reproduces key BBB markers and predicts compound permeability across the BBB in vivo. Here, we describe incorporating human GBM stem cells (GSC) into the organoid to form BTB organoids, presenting a novel scalable multicellular human brain tumor organoid model that simulates the BTB to facilitate therapeutic development in neuro-oncology. This model involves the self-assembly of essential BTB components, including human brain microvascular endothelial cells, astrocytes, brain pericytes and patient-derived GSCs, co-cultured under ultralow attachment conditions. Immunofluorescence microscopy results show reduced ZO-1 and Mfsd2a expression in the BTB compared to the control BBB organoids, indicating tight junction disruption and BBB breakdown by the GSCs. Correspondingly, we observe increased dextran permeability in the BTB organoids compared to the control BBB organoids, which is dependent on the GSC count per organoid. Electron and confocal microscopy reveal disorganized neovascular structures and invasive GSC behavior in the BTB organoids. Lastly, using a known GBM-targeting peptide (BTP-7), as well as a known BBB-penetrating adeno-associated virus (AAV.CPP.16) as proofs-of-concept, we demonstrate the efficacy of using the BTB organoids for modeling therapeutic delivery to brain tumors. In summary, the BTB organoid platform provides a tractable in vitro system reflecting the brain tumor microenvironment, enabling the study of GBM cell properties, investigation of barrier behavior, and modeling therapeutic delivery to brain tumors.

Study on the effects of different anti-VEGF drugs on fibrovascular membranes of proliferative diabetic retinopathy

PurposeTo investigate the effects of different anti-VEGF drugs on fibrovascular membranes (FVM) in proliferative diabetic retinopathy (PDR). In addition, in vitro model was used to simulate the intraocular fibroblasts barrier to explore the penetration of different anti-VEGF drugs. Methods24 eyes from 24 PDR patients with FVM were recruited for this prospective observational study. The patients were randomized to receive one of three anti-VEGF drugs (Ranibizumab, Conbercept, or Aflibercept). Then neovascular structures were assessed by optical coherence tomography angiography (OCTA) before intravitreal injection (pre-IVT) and 1, 2, and 3 days after intravitreal injection (post-IVT). The changes in vessels area (VSA), vessels percentage area (VPA), junction density (JD), and average lacunarity (AL) were analyzed by using the image processing software Angiotool. In vitro penetrating model with fibroblasts barrier was used to compare the effects of the three drugs on human retinal vascular endothelial cells (HRVECs) over 3 days by Cell proliferation measurement. Moreover, the drug concentrations in the penetrating model were detected by liquid chromatography-mass spectrometry (LC-MS). ResultsThe VSA, VPA, and JD all decreased, while the AL increased in Ranibizumab group(n = 8), Conbercept group (n = 8), and Aflibercept group (n = 8) within 3 days (P<0.05). Meanwhile, under the condition of the same amount of substance, the inhibition effect of Ranibizumab on HRVEC was the strongest in the penetrating model evaluated by CCK8 absorbance experiments of HRVECs (FCCK8=6.493, PCCK8= 0.0051), and the number of transmembrane molecules in the Ranibizumab group was also the largest within 3 days (F = 8.209, P = 0.0006) among the three groups. ConclusionAngiotool is feasible to reconstruct the neovascular structure on the FVM in OCTA images. The three different anti-VEGF drugs can significantly reduce the vascular area and density on the proliferating membranes, and there is no significant difference in the anti-neovascularization among the three drugs clinically. However, small molecule drug is more penetrating and move faster across membranes in vitro cell model. Clinical Trial RegistrationThis trial is registered with the Chinese Clinical Trial Registry (http://www.chictr.org.cn, registration number ChiCTR2300067476).

Current and Novel Therapeutic Approaches for Treatment of Neovascular Age-Related Macular Degeneration.

Age-related macular degeneration AMD is one of the leading causes of blindness in the elderly population. An advanced form of AMD known as neovascular AMD (nAMD) is implicated as the main attributor of visual loss among these patients. The hallmark feature of nAMD is the presence of neovascular structures known as choroidal neovascular membranes (CNVs), along with fluid exudation, hemorrhages, and subretinal fibrosis. These pathological changes eventually result in anatomical and visual loss. A type of proangiogenic factor known as vascular endothelial growth factor (VEGF) has been known to mediate the pathological process behind nAMD. Therefore, therapy has transitioned over the years from laser therapy that ablates the lesions to using Anti-VEGF to target the pathology directly. In this work, we provide an overview of current and emerging therapies for the treatment of nAMD. Currently approved Anti-VEGF agents include ranibizumab, aflibercept, and brolucizumab. Bevacizumab, also an Anti-VEGF agent, is used to manage nAMD even though this is an off-label use. While Anti-VEGF agents have provided a favorable prognosis for nAMD, they are associated with a substantial financial burden for patients and the healthcare system, due to their high cost as well as the need for frequent repeat treatments and visits. Emerging therapies and studies aim to extend the intervals between required treatments and introduce new treatment modalities that would improve patients' compliance and provide superior results.

Abstract P2023: Non-cardiomyocytes From Chronic Unloaded Post LVAD Heart Form Organized Complex Neovascular Structures When Compared To Those From Heart Failure.

Introduction: Chronic unloading of the left ventricle (LV) has shown increased endothelial density suggestive of a vascular recovery. Mechanisms of such neovascularization have not been fully explored. Hypothesis: Non-myocyte transitions may play a role in neovascularization in cardiac recovery. Methods: We utilized fresh cardiac tissue obtained from patients at the time of LVAD (pre-LVAD core - representing HF) and from a LVAD patient at the time of transplant (Post-LVAD-representing chronic unloaded heart). Using disaggregation, cardiac non-myocytes were isolated, and the resulting pellet of cardiac non-myocytes was divided and plated in both fibroblast (FGM) and endothelial (EGM) growth media. Results: Cells from post-LVAD patient in EGM (grown on glass coverslip) showed a complex organizational network with the presence of CD31+ cells in vessel like structures surrounded by vimentin positive cells (See Figure). These networks were not observed in any of the other culture conditions. We also observed that cells from post-LVAD grew much faster compared to pre-LVAD cells. Conclusions: To our knowledge, this is the first-time disaggregated cells from human heart samples have been studied in culture. These preliminary observations support the ability of non-cardiomyocytes in a chronic unloaded and rested heart to form neovascular structures not seen in cells from the diseased state of heart failure. This platform can serve to further understand the behavior and role of complex non-myocyte interactions in cardiac recovery.

A novel method for visualizing and tracking endogenous mRNA in a specific cell population in pathological neovascularization

Diabetic retinopathy, retinopathy of prematurity and retinal vein occlusion are potentially blinding conditions largely due to their respective neovascular components. The development of real-time in vivo molecular imaging methods, to assess levels of retinal neovascularization (NV), would greatly benefit patients afflicted with these conditions. mRNA hybridization techniques offer a potential method to image retinal NV. The success of these techniques hinges on the selection of a target mRNA whose tissue levels and spatial expression patterns correlate closely with disease burden. Using a model of oxygen-induced retinopathy (OIR), we previously observed dramatic increases in retinal endoglin that localized to neovascular structures (NV), directly correlating with levels of neovascular pathology. Based on these findings, we have investigated Endoglin mRNA as a potential marker for imaging retinal NV in OIR mice. Also of critical importance, is the application of innovative technologies capable of detecting mRNAs in living systems with high sensitivity and specificity. To detect and visualize endoglin mRNA in OIR mice, we have designed and synthesized a novel imaging probe composed of short-hairpin anti-sense (AS) endoglin RNA coupled to a fluorophore and black hole quencher (AS-Eng shRNA). This assembly allows highly sensitive fluorescence emission upon hybridization of the AS-Eng shRNA to cellular endoglin mRNA. The AS-Eng shRNA is further conjugated to a diacyl-lipid (AS-Eng shRNA–lipid referred to as probe). The lipid moiety binds to serum albumin facilitating enhanced systemic circulation of the probe. OIR mice received intraperitoneal injections of AS-Eng shRNA–lipid. Ex vivo imaging of their retinas revealed specific endoglin mRNA dependent fluorescence superimposed on neovascular structures. Room air mice receiving AS-Eng shRNA–lipid and OIR mice receiving a non-sense control probe showed little fluorescence activity. In addition, we found that cells in neovascular lesions labelled with endoglin mRNA dependent fluorescence, co-labelled with the macrophage/microglia-associated marker IBA1. Others have shown that cells expressing macrophage/microglia markers associate with retinal neovascular structures in proportion to disease burden. Hence we propose that our probe may be used to image and to estimate the levels of retinal neovascular disease in real-time in living systems.

Antibody-based delivery of interleukin-9 to neovascular structures: Therapeutic evaluation in cancer and arthritis.

Interleukin-9 is a cytokine with multiple functions, including the ability to activate group 2 innate lymphoid cells, which has been postulated to be therapeutically active in mouse models of arthritis. Similarly, interleukin-9 has been suggested to play an important role in tumor immunity. Here, we describe the cloning, expression, and characterization of three fusion proteins based on murine interleukin-9 and the F8 antibody, specific to the alternatively spliced EDA domain of fibronectin. EDA is strongly expressed in cancer and in various arthritic conditions, while being undetectable in the majority of healthy organs. Interleukin-9-based fusion proteins with an irrelevant antibody specific to hen egg lysozyme served as negative control in our study. The fusion proteins were characterized by quantitative biodistribution analysis in tumor-bearing mice using radioiodinated protein preparations. The highest tumor uptake and best tumor:organ ratios were observed for a format, in which the interleukin-9 moiety was flanked by two units of the F8 antibody in single-chain Fv format. Biological activity of interleukin-9 was retained when the payload was fused to antibodies. However, the targeted delivery of interleukin-9 to the disease site resulted in a modest anti-tumor activity in three different murine models of cancer (K1735M2, CT26, and F9), while no therapeutic benefit was observed in a collagen induced model of arthritis. Collectively, these results confirm the possibility to deliver interleukin-9 to the site of disease but cast doubts about the alleged therapeutic activity of this cytokine in cancer and arthritis, which has been postulated in previous publications.

Influence of porcine urinary bladder matrix and porcine acellular dermal matrix on wound healing of full-thickness skin defect in diabetic mice

Influence of porcine urinary bladder matrix and porcine acellular dermal matrix on wound healing of full-thickness skin defect in diabetic mice

VASCULAR COMPLEXITY ANALYSIS IN OPTICAL COHERENCE TOMOGRAPHY ANGIOGRAPHY OF DIABETIC RETINOPATHY.

This study aimed to verify the feasibility of using vascular complexity features for objective differentiation of controls and nonproliferative diabetic retinopathy (NPDR) and proliferative diabetic retinopathy (PDR) patients. This was a cross-sectional study conducted in a tertiary, subspecialty, academic practice. The cohort included 20 control subjects, 60 NPDR patients, and 56 PDR patients. Three vascular complexity features, including the vessel complexity index, fractal dimension, and blood vessel tortuosity, were derived from each optical coherence tomography angiography image. A shifting-window measurement was further implemented to identify local feature distortions due to localized neovascularization and mesh structures in PDR. With mean value analysis of the whole-image, only the vessel complexity index and blood vessel tortuosity were able to classify NPDR versus PDR patients. Comparative shifting-window measurement revealed increased sensitivity of complexity feature analysis, particularly for NPDR versus PDR classification. A multivariate regression model indicated that the combination of all three vascular complexity features with shifting-window measurement provided the best classification accuracy for controls versus NPDR versus PDR. Vessel complexity index and blood vessel tortuosity were the most sensitive in differentiating NPDR and PDR patients. A shifting-window measurement increased the sensitivity significantly for objective optical coherence tomography angiography classification of diabetic retinopathy.

Inferior and Intra-/Peri-Articular Superior Sacroiliac Joint Injection Approaches Under Ultrasound Guidance to Treat Metastasis-Related Posterior Pelvic Bone Pain.

Numerous mechanical and pathologic variables contribute to sacroiliac joint (SIJ) pain. The oncologic population has additional considerations, including tumor burden causing fracture, nerve compression, joint instability, and periosteal inflammation. Post-treatment changes may also restrict joint mobility, causing transitional pain. Currently, fluoroscopically guided SIJ injections, aimed at the inferior one third of the SIJ, are the gold standard for treatment but have only been described in the nononcologic population. Ultrasound (US) guidance may confer several benefits, including positioning, ease of procedure, lower costs, and, importantly, guidance to avoid neovascularization, metastatic disease, and other soft tissue structures. We aim to describe the advantages of US-guided SIJ injections for refractory malignant SIJ pain from extra-articular tumors. We then describe our technique and decision framework for accessing the superior or inferior SIJ in patients with metastatic sacroiliac pain. A retrospective review was performed on 5 patients with refractory malignant SIJ pain who underwent US-guided superior or inferior approach SIJ injection. Using imaging and outcomes, we developed a decision framework. Patients received either inferior or superior approach SIJ injections depending on location of tumor, extent of tumor invasion, and stability of the SIJ as per our framework. All patients reported improvement in pain and function without complications. We propose a decision framework for inferior vs. superior approach US-guided SIJ injections in the oncologic population with SIJ pain from metastases to the pelvis or sacrum. Having multiple techniques to approach the SIJ is important in the oncologic population, in whom metastatic tumor burden poses a technical challenge to performing these injections.

Three-dimensional pattern of extraretinal neovascular development in retinopathy of prematurity

The application of three-dimensional (3D) visualization techniques to evaluate the earliest visible onset of abnormal retinal vascular development in preterm infants with retinopathy of prematurity (ROP), using bedside non-contact optical coherence tomography (OCT) imaging to characterize morphology and sequential structural changes of abnormal extraretinal neovascularization. Thirty-one preterm infants undergoing routine ROP screening with written informed consent for research imaging were enrolled in this prospective observational study. We imaged the macula and temporal periphery of preterm infants using a handheld OCT system (Envisu 2300 or handheld swept-source research system). The scans obtained were segmented and, using enhanced ray casting, were converted to 3D volumes to which color filter was applied. Using colorized 3D visualization, we defined extraretinal neovascular structures as buds, bridging networks, and placoid lesions. We could longitudinally follow progression and regression of extraretinal neovascularization in stage 3 ROP after treatment in one infant over 12weeks and document the appearance of early buds, and formation of florid neovascularization. From stages 2 to 3 ROP, we observed progression from sessile buds to a complex plaque that corresponded to stage 3 ROP on clinical examination. We demonstrated regression of neovascular complexes to small pre-retinal tufts after treatment with anti-VEGF. The extension of OCT processing to include surface flattening and colorization that further improved structural analysis rendered better understanding of extraretinal tissue. Our ability to image similar areas in the same infant over multiple visits enabled us to study the evolution of these structural components and follow pathological vascular events longitudinally in development and regression after treatment. These methods can be applied to further study which are likely contribute to our understanding of the pathophysiology of neovascularization in ROP.

Angiopoietin-1 Promotes the Integrity of Neovascularization in the Subcutaneous Matrigel of Type 1 Diabetic Rats.

Objective This study aimed to investigate the effects of Ang-1 on neovascularization of diabetic organs by subcutaneous Matrigel angiogenesis model, established in type 1 diabetic rats. Methods Ang-1 adenoviral vector was constructed. The rat model was established by STZ and divided into four group. The Matrigel was inserted subcutaneously into the abdominal cavity of rats at 8 weeks, the treatment group was injected with Ang-1 adenovirus vector via tail vein, and the rats were sacrificed at 10 weeks. Neovascularization of Matrigel was observed with transmission electron microscopy. The marker of vascular endothelial cell and pericyte were detected by immunofluorescence. Immunohistochemical detection of the neovascular endothelial junction protein was performed. RT-PCR was used to determine protein expression of neovascular in Matrigel. Results Vascular cavity-like structure could be seen in subcutaneous Matrigel of diabetic rats, and the cavity was filled with a lot of red blood cells. Transmission electron microscopy showed that neovascular endothelial structure of the Matrigel was incomplete, while the Ang-1 treatment group had more vascular cavity-like structures, intact vascular endothelial structure, and reduced inflammatory cell infiltration in Matrigel. Additionally, the integrity of vascularization improved, and the marker of pericyte and the cell tight junctions protein was upregulated in Ang-1 treatment group. Conclusion Hyperglycemia could induce pathological angiogenesis in subcutaneous Matrigel of diabetic rats, and Ang-1 could upregulate the expression of intercellular junction protein in subcutaneous Matrigel of diabetic rats and promote the integrity of neovascularization in the subcutaneous Matrigel of diabetic rats.

Retinal Lesion Detection With Deep Learning Using Image Patches.

PurposeTo develop an automated method of localizing and discerning multiple types of findings in retinal images using a limited set of training data without hard-coded feature extraction as a step toward generalizing these methods to rare disease detection in which a limited number of training data are available.MethodsTwo ophthalmologists verified 243 retinal images, labeling important subsections of the image to generate 1324 image patches containing either hemorrhages, microaneurysms, exudates, retinal neovascularization, or normal-appearing structures from the Kaggle dataset. These image patches were used to train one standard convolutional neural network to predict the presence of these five classes. A sliding window method was used to generate probability maps across the entire image.ResultsThe method was validated on the eOphta dataset of 148 whole retinal images for microaneurysms and 47 for exudates. A pixel-wise classification of the area under the curve of the receiver operating characteristic of 0.94 and 0.95, as well as a lesion-wise area under the precision recall curve of 0.86 and 0.64, was achieved for microaneurysms and exudates, respectively.ConclusionsRegionally trained convolutional neural networks can generate lesion-specific probability maps able to detect and distinguish between subtle pathologic lesions with only a few hundred training examples per lesion.

Mixed type cutaneous haemangiosarcoma in a dog

A six year old Labrador retriever with the history of growth over the skin of submandibular region was presented. The growth was removed and subjected for histopathological study. The tissue revealed variable sized vascular channels filled with blood. The vascular channels were lined by pleomorphic cells. The stroma revealed compact pleomorphic, hyperchromatic spindle shaped cells with prominent nucleoli and mitotic figures. The neoplastic cells forming neovascular structures originated from vascular endothelial cells. The mass composed of both cavernous and solid type of haemangiosarcoma. Based on the histopathological features, the case was diagnosed as cutaneous haemangiosarcoma with a mixture of cavernous and solid type in a Labrador retriever.

Development of Endothelial Cell Networks in 3D Tissues by Combination of Melt Electrospinning Writing with Cell-Accumulation Technology.

A remaining challenge in tissue engineering approaches is the in vitro vascularization of engineered constructs or tissues. Current approaches in engineered vascularized constructs are often limited in the control of initial vascular network geometry, which is crucial to ensure full functionality of these constructs with regard to cell survival, metabolic activity, and potential differentiation ability. Herein, the combination of 3D-printed poly-ε-caprolactone scaffolds via melt electrospinning writing with the cell-accumulation technique to enable the formation and control of capillary-like network structures is reported. The cell-accumulation technique is already proven itself to be a powerful tool in obtaining thick (50 µm) tissues and its main advantage is the rapid production of tissues and its ease of performance. However, the applied combination yields tissue thicknesses that are doubled, which is of outstanding importance for an improved handling of the scaffolds and the generation of clinically relevant sample volumes. Moreover, a correlation of increasing vascular endothelial growth factor secretion to hypoxic conditions with increasing pore sizes and an assessment of the formation of neovascular like structures are included.

Human ex vivo model of iris angiogenesis

PurposeTo assess iris angiogenesis using human explants and determine the role of hypoxia during iris neovascularization.MethodsHuman irises were isolated post‐mortem from human research consented donnors. Explants were seeded onto extracellular matrix (ECM), and kept at normal oxygen concentrations (normoxia, 20% O2) or exposed to hypoxia (1% O2) in standard tissue culture conditions for 48 h. Iris angiogenesis was analyzed by quantifying the neovascular area. Furthermore, iris neovascular structures were characterized by immunofluorescence.ResultsHuman iris ex vivo neoangiogenesis could be observed within 48 h after the explants were seeded onto ECM, and was defined as vascular sprouts and/or tufts. Vascular sprouts were more common in normoxia, while vascular tufts were mostly formed in hypoxia. A significant increase in vascular area was observed in irises exposed to hypoxia when compared to normoxia. Positive immunoreactivity to specific endothelial cell markers confirmed human iris neoangiogenesis ex vivo.ConclusionsOur data shows the first human ex vivo neoangiogenesis assay, using iris. The method here presented could have potential use in testing pro‐ and anti‐angiogenic drugs in an human ocular context.

Impact of adipose tissue or umbilical cord derived mesenchymal stem cells on the immunogenicity of human cord blood derived endothelial progenitor cells.

The application of autologous endothelial progenitor cell (EPC) transplantation is a promising approach in therapeutic cardiovascular diseases and ischemic diseases. In this study, we compared the immunogenicity of EPCs, adipose tissue (AD)-derived mesenchymal stem cells (MSCs) and umbilical cord (UC)-derived MSCs by flow cytometry and the mixed lymphocyte reaction. The impact of AD-MSCs and UC-MSCs on the immunogenicity of EPCs was analyzed by the mixed lymphocyte reaction and cytokine secretion in vitro and was further tested by allogenic peripheral blood mononuclear cell (PBMC) induced immuno-rejection on a cell/matrigel graft in an SCID mouse model. EPCs and AD-MSCs express higher levels of MHC class I than UC-MSCs. All three kinds of cells are negative for MHC class II. UC-MSCs also express lower levels of IFN-γ receptor mRNA when compared with EPCs and AD-MSCs. EPCs can stimulate higher rates of proliferation of lymphocytes than AD-MSCs and UC-MSCs. Furthermore, AD-MSCs and UC-MSCs can modulate immune response and inhibit lymphocyte proliferation induced by EPCs, mainly through inhibition of the proliferation of CD8+ T cells. Compared with UC-MSCs, AD-MSCs can significantly improve vessel formation and maintain the integrity of neovascular structure in an EPC+MSC/matrigel graft in SCID mice, especially under allo-PBMC induced immuno-rejection. In conclusion, our study shows that AD-MSC is a powerful candidate to minimize immunological rejection and improve vessel formation in EPC transplantation treatment.

Retinal blood vessel extraction using tunable bandpass filter and fuzzy conditional entropy

Background and objectivesExtraction of blood vessels on retinal images plays a significant role for screening of different opthalmologic diseases. However, accurate extraction of the entire and individual type of vessel silhouette from the noisy images with poorly illuminated background is a complicated task. To this aim, an integrated system design platform is suggested in this work for vessel extraction using a sequential bandpass filter followed by fuzzy conditional entropy maximization on matched filter response. MethodsAt first noise is eliminated from the image under consideration through curvelet based denoising. To include the fine details and the relatively less thick vessel structures, the image is passed through a bank of sequential bandpass filter structure optimized for contrast enhancement. Fuzzy conditional entropy on matched filter response is then maximized to find the set of multiple optimal thresholds to extract the different types of vessel silhouettes from the background. Differential Evolution algorithm is used to determine the optimal gain in bandpass filter and the combination of the fuzzy parameters. Using the multiple thresholds, retinal image is classified as the thick, the medium and the thin vessels including neovascularization. ResultsPerformance evaluated on different publicly available retinal image databases shows that the proposed method is very efficient in identifying the diverse types of vessels. Proposed method is also efficient in extracting the abnormal and the thin blood vessels in pathological retinal images. The average values of true positive rate, false positive rate and accuracy offered by the method is 76.32%, 1.99% and 96.28%, respectively for the DRIVE database and 72.82%, 2.6% and 96.16%, respectively for the STARE database. Simulation results demonstrate that the proposed method outperforms the existing methods in detecting the various types of vessels and the neovascularization structures. ConclusionsThe combination of curvelet transform and tunable bandpass filter is found to be very much effective in edge enhancement whereas fuzzy conditional entropy efficiently distinguishes vessels of different widths.

Abstract SY35-04: Arming tumor-targeting antibodies with cytokine payloads: Emerging clinical results

Abstract Antibodies can be used to deliver bioactive molecules (drugs, cytokines, photosensitizers, radionuclides, etc.) to the tumor environment, thus sparing normal tissues. Pharmacodelivery strategies targeting certain modified extracellular matrix components, associated with the tumor neo-vasculature, is particularly attractive, because of: (i) the abundance and stability of these antigens (e.g., splice isoforms of fibronectin and tenascin-C); (ii) the dependence of cancer on new blood vessels; (iii) the accessibility of neo-vascular structures for therapeutic agents coming from the blood-stream; (iv) the fact that some extracellular matrix components are very abundant in many different cancer types, while being virtually undetectable in most normal adult tissues [Refs. 1-8]. In this lecture, I will present our experience (at a preclinical and clinical level) in the development and clinical applications of antibody-cytokine fusion proteins (“immunocytokines”) for cancer therapy applications. In particular, I will focus on pro-inflammatory immunocytokines specific to the alternatively-spliced EDA and EDB domains of fibronectin, or A1 domain of tenascin-C, used either as single agents or in combination. Immunocytokines based on IL2 or TNF as payloads are able to dramatically increase the density of certain leukocytes (e.g., T cells and NK cells) at the neoplastic site in various types of malignancies.